Oxidized lipids and uses thereof in the treatment of inflammatory diseases and disorders

ABSTRACT

Novel synthetic oxidized lipids and methods utilizing oxidized lipids for treating and preventing an inflammation associated with an endogenous oxidized lipid are provided.

RELATED APPLICATIONS

This application is a divisional of pending U.S. patent application Ser.No. 11/528,657, filed on Sep. 28, 2006, which is a continuation-in-partof U.S. patent application Ser. No. 10/567,543, filed on Apr. 25, 2008,which is a National Phase of PCT Patent Application No.PCT/IL2004/000453, filed on May 27, 2004, which is a Continuation ofU.S. patent application Ser. No. 10/445,347, filed on May 27, 2003, nowU.S. Pat. No. 6,838,452 issued on Jan. 4, 2005. The contents of theabove applications are all incorporated by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to novel oxidized lipids and to methodsemploying oxidized lipids for treating or preventing an inflammationassociated with endogenous oxidized lipids. The methods of the presentinvention can be utilized in treating or preventing inflammationassociated diseases and disorders such as, for example, atherosclerosisand related disorders, autoimmune diseases or disorders, andproliferative disease or disorders.

Cardiovascular disease is a major health risk throughout theindustrialized world. Atherosclerosis, the most prevalent ofcardiovascular diseases, is the principal cause of heart attack, stroke,and gangrene of the extremities, and as such, the principle cause ofdeath in the United States. Atherosclerosis is a complex diseaseinvolving many cell types and molecular factors (for a detailed review,see Ross, 1993, Nature 362: 801-809). The process, which occurs inresponse to insults to the endothelium and smooth muscle cells (SMCs) ofthe wall of the artery, consists of the formation of fibrofatty andfibrous lesions or plaques, preceded and accompanied by inflammation.Plaque destabilization may lead to further complications such as ruptureand thrombosis, which result from an excessiveinflammatory-fibroproliferative response to numerous different forms ofinsults. For example, shear stresses are thought to be responsible forthe frequent occurrence of atherosclerotic plaques in regions of thecirculatory system where turbulent blood flow occurs, such as branchpoints and irregular structures.

The first observable event in the formation of an atherosclerotic plaqueoccurs when inflammatory cells such as monocyte-derived macrophagesadhere to the vascular endothelial layer and transmigrate through to thesub-endothelial space. Elevated plasma LDL levels lead to lipidengorgement of the vessel walls, with adjacent endothelial cellsproducing oxidized low density lipoprotein (LDL). In addition,lipoprotein entrapment by the extracellular matrix leads to progressiveoxidation of LDL by lipoxygenases, reactive oxygen species,peroxynitrite and/or myeloperoxidase. These oxidized LDL's are thentaken up in large amounts by monocytes through scavenger receptorsexpressed on their surfaces.

Lipid-filled monocytes and smooth-muscle derived cells (SMCs) are calledfoam cells, and are the major constituent of the fatty streak.Interactions between foam cells, endothelial cells and smooth musclecells surrounding them produce a state of chronic local inflammationwhich can eventually lead to activation of endothelial cells, increasedmacrophage apoptosis, smooth muscle cell proliferation and migration,and the formation of a fibrous plaque (Hajjar, D P and Haberland, M E,J. Biol Chem 1997 Sep. 12; 272(37):22975-78). Plaque rupture andthrombosis occlude the blood vessels concerned and thus restrict theflow of blood, resulting in ischemia, a condition characterized by alack of oxygen supply in tissues of organs due to inadequate perfusion.When the involved arteries block the blood flow to the heart, a personis afflicted with a ‘heart attack’; when the brain arteries occlude, theperson experiences a stroke. When arteries to the limbs narrow, theresult is severe pain, decreased physical mobility and possibly the needfor amputation.

Oxidized LDL has been implicated in the pathogenesis of atherosclerosisand atherothrombosis, by its action on monocytes and smooth musclecells, and by inducing endothelial cell apoptosis, impairinganticoagulant balance in the endothelium. Oxidized LDL also inhibitsanti-atherogenic HDL-associated breakdown of oxidized phospholipids(Mertens, A and Holvoet, P, FASEB J 2001 October; 15(12):2073-84). Thisassociation is also supported by many studies demonstrating the presenceof oxidized LDL in the plaques in various animal models of atherogenesisand the retardation of atherogenesis through inhibition of oxidation bypharmacological and/or genetic manipulations (see, for example, WitztumJ and Steinberg, D, Trends Cardiovasc Med 2001 April-May; 11(3-4):93-102for a review of current literature). Indeed, oxidized LDL andmalondialdehyde (MDA)-modified LDL have been recently proposed asaccurate blood markers for 1^(st) and 2^(nd) stages of coronary arterydisease (U.S. Pat. Nos. 6,309,888, to Holvoet et al., and 6,255,070 toWitztum, et al.).

Reduction of LDL oxidation and activity has been the target of a numberof suggested clinical applications for treatment and prevention ofcardiovascular disease. Bucala, et al. (U.S. Pat. No. 5,869,534)discloses methods for the modulation of lipid peroxidation by reducingadvanced glycosylation end product, lipid characteristic of age-,disease- and diabetes-related foam cell formation. Tang et al., atIncyte Pharmaceuticals, Inc. (U.S. Pat. No. 5,945,308) have disclosedthe identification and proposed clinical application of a Human OxidizedLDL Receptor in the treatment of cardiovascular and autoimmune diseasesand cancer.

Atherosclerosis and Autoimmune Disease

Because of the presumed role of the excessiveinflammatory-fibroproliferative response in atherosclerosis andischemia, a growing number of researchers have attempted to define anautoimmune component of vascular injury. In autoimmune diseases theimmune system recognizes and attacks normally non-antigenic bodycomponents (autoantigens), in addition to attacking invading foreignantigens. The autoimmune diseases are classified as auto- (or self-)antibody mediated or cell mediated diseases. Typical autoantibodymediated autoimmune diseases are myasthenia gravis and idiopathicthrombocytopenic purpura (ITP), while typical cell mediated diseases areHashimoto's thyroiditis and type I (Juvenile) Diabetes.

The recognition that immune mediated processes prevail withinatherosclerotic lesions stemmed from the consistent observation oflymphocytes and macrophages in the earliest stages, namely the fattystreaks. These lymphocytes which include a predominant population ofCD4+ cells (the remainder being CD8+ cells) were found to be moreabundant over macrophages in early lesions, as compared with the moreadvanced lesions, in which this ratio tends to reverse. These findingsposed questions as to whether they reflect a primary immunesensitization to a possible antigen or alternatively stand as a mereepiphenomenon of a previously induced local tissue damage. Regardless ofthe factors responsible for the recruitment of these inflammatory cellsto the early plaque, they seem to exhibit an activated state manifestedby concomitant expression of MHC class II HLA-DR and interleukin (IL)receptor as well as leukocyte common antigen (CD45R0) and the very lateantigen 1 (VLA-1) integrin.

The on-going inflammatory reaction in the early stages of theatherosclerotic lesion may either be the primary initiating eventleading to the production of various cytokines by the local cells (i.e.endothelial cells, macrophages, smooth muscle cells and inflammatorycells), or it may be that this reaction is a form of the body's defenseimmune system towards the hazardous process. Some of the cytokines whichhave been shown to be upregulated by the resident cells include TNF-α,IL-1, IL-2, IL-6, IL-8, IFN-γ and monocyte chemoattractant peptide-1(MCP-1). Platelet derived growth factor (PDGF) and insulin-like growthfactor (IGF) which are expressed by all cellular constituents withinatherosclerotic plaques have also been shown to be overexpressed, thuspossibly intensifying the preexisting inflammatory reaction by aco-stimulatory support in the form of a mitogenic and chemotacticfactor. Recently, Uyemura et al. (Cross regulatory roles of IL-12 andIL-10 in atherosclerosis. J Clin Invest 1996 97; 2130-2138) haveelucidated type 1 T-cell cytokine pattern in human atheroscleroticlesions exemplified by a strong expression of IFN-γ but not IL-4 mRNA incomparison with normal arteries. Furthermore, IL-12- a T-cell growthfactor produced primarily by activated monocytes and a selective inducerof Th1 cytokine pattern, was found to be overexpressed within lesions asmanifested by the abundance of its major heterodimer form p70 and p40(its dominant inducible protein) mRNA.

Similar to the strong evidence for the dominance of the cellular immunesystem within the atherosclerotic plaque, there is also ample datasupporting the involvement of the local humoral immune system. Thus,deposition of immunoglobulins and complement components have been shownin the plaques in addition to the enhanced expression of the C3b andC3Bi receptors in resident macrophages.

Valuable clues with regard to the contribution of immune mediatedinflammation to the progression of atherosclerosis come from animalmodels. Immunocompromised mice (class I MHC deficient) tend to developaccelerated atherosclerosis as compared with immune competent mice.Additionally, treatment of C57BL/6 mice (Emeson E E, Shen M L.Accelerated atherosclerosis in hyperlipidemic C57BL/6 mice treated withcyclosporin A. Am J Pathol 1993; 142: 1906-1915) and New-Zealand Whiterabbits (Roselaar S E, Schonfeld G, Daugherty A. Enhanced development ofatherosclerosis in cholesterol fed rabbits by suppression of cellmediated immunity. J Clin Invest 1995; 96: 1389-1394) with cyclosporinA, a potent suppressor of IL-2 transcription resulted in a significantlyenhanced atherosclerosis under “normal” lipoprotein “burden”. Theselatter studies may provide insight into the possible roles of the immunesystem in counteracting the self-perpetuating inflammatory processwithin the atherosclerotic plaque.

Atherosclerosis is not a classical autoimmune disease, although some ofits manifestations such as the production of the plaque which obstructsthe blood vessels may be related to aberrant immune responsiveness. Inclassical autoimmune disease, one can often define very clearly thesensitizing autoantigen attacked by the immune system and thecomponent(s) of the immune system which recognize the autoantigen(humoral, i.e. autoantibody or cellular, i.e. lymphocytes). Above all,one can show that by passive transfer of these components of the immunesystem the disease can be induced in healthy animals, or in the case ofhumans the disease may be transferred from a sick pregnant mother to heroffspring. Many of the above are not prevailing in atherosclerosis. Inaddition, the disease definitely has common risk factors such ashypertension, diabetes, lack of physical activity, smoking and others,the disease affects elderly people and has a different geneticpreponderance than in classical autoimmune diseases.

Treatment of autoimmune inflammatory disease may be directed towardssuppression or reversal of general and/or disease-specific immunereactivity. Thus Aiello, for example (U.S. Pat. Nos. 6,034,102 and6,114,395) discloses the use of estrogen-like compounds for treatmentand prevention of atherosclerosis and atherosclerotic lesion progressionby inhibition of inflammatory cell recruitment. Similarly, Medford etal. (U.S. Pat. No. 5,846,959) disclose methods for the prevention offormation of oxidized PUFA, for treatment of cardiovascular andnon-cardiovascular inflammatory diseases mediated by the cellularadhesion molecule VCAM-1. Furthermore, Falb (U.S. Pat. No. 6,156,500)designates a number of cell signaling and adhesion molecules abundant inatherosclerotic plaque and disease as potential targets ofanti-inflammatory therapies.

Since oxidized LDL has been clearly implicated in the pathogenesis ofatherosclerosis (see above), the contribution of these prominent plaquecomponents to autoimmunity in atheromatous disease processes has beeninvestigated.

Immune responsiveness to Oxidized LDL It is known that oxidized LDL (OxLDL) is chemotactic for T-cells and monocytes. Ox LDL and its byproductsare also known to induce the expression of factors such as monocytechemotactic factor 1, secretion of colony stimulating factor andplatelet activating properties, all of which are potent growthstimulants.

The active involvement of the cellular immune response inatherosclerosis has recently been substantiated by Stemme S., et al.(Proc Natl Acad Sci USA 1995; 92: 3893-97), who isolated CD4+ withinplaques clones responding to Ox LDL as stimuli. The clones correspondingto Ox LDL (4 out of 27) produced principally interferon-γ rather thanIL-4. It remains to be seen whether the above T-cell clones representmere contact with the cellular immune system with the inciting strongimmunogen (Ox LDL) or that this reaction provides means of combating theapparently indolent atherosclerotic process.

The data regarding the involvement of the humoral mechanisms and theirmeaning are much more controversial. One recent study reported increasedlevels of antibodies against MDA-LDL, a metabolite of LDL oxidation, inwomen suffering from heart disease and/or diabetes (Dotevall, et al.,Clin Sci 2001 November; 101(5): 523-31). Other investigators havedemonstrated antibodies recognizing multiple epitopes on the oxidizedLDL, representing immune reactivity to the lipid and apolipoproteincomponents (Steinerova A., et al., Physiol Res 2001; 50(2): 131-41) inatherosclerosis and other diseases, such as diabetes, renovascularsyndrome, uremia, rheumatic fever and lupus erythematosus. Severalreports have associated increased levels of antibodies to Ox LDL withthe progression of atherosclerosis (expressed by the degree of carotidstenosis, severity of peripheral vascular disease etc.). Most recently,Sherer et al. (Cardiology 2001; 95(1):20-4) demonstrated elevated levelsof antibodies to cardiolipin, beta 2GPI and OxLDL, in coronary heartdisease. Thus, there seems to be a consensus as to the presence of OxLDL antibodies in the form of immune complexes within atheroscleroticplaque, although the true significance of this finding has not beenestablished.

Antibodies to Ox LDL have been hypothesized as playing an active role inlipoprotein metabolism. Thus, it is known that immune complexes of OxLDL and its corresponding antibodies are taken up more efficiently bymacrophages in suspension as compared with Ox LDL. No conclusions can bedrawn from this consistent finding on the pathogenesis ofatherosclerosis since the question of whether the accelerated uptake ofOx LDL by the macrophages is beneficial or deleterious has not yet beenresolved.

Important data as to the significance of the humoral immune system inatherogenesis comes from animal models. It has been found thathyperimmunization of LDL-receptor deficient rabbits with homologousoxidized LDL, resulted in the production of high levels of anti-Ox LDLantibodies and was associated with a significant reduction in the extentof atherosclerotic lesions as compared with a control group exposed tophosphate-buffered saline (PBS). A decrease in plaque formation has alsobeen accomplished by immunization of rabbits with cholesterol richliposomes with the concomitant production of anti-cholesterolantibodies, yet this effect was accompanied by a 35% reduction in verylow density lipoprotein cholesterol levels.

Thus, both the pathogenic role of oxidized LDL components and theirimportance as autoantigens in atherosclerosis, as well as otherdiseases, have been extensively demonstrated in laboratory and clinicalstudies.

Mucosal-Mediated Immunomodulation in Treatment of Autoimmune Disease

Recently, new methods and pharmaceutical formulations have been foundthat are useful for treating autoimmune diseases (and related T-cellmediated inflammatory disorders such as allograft rejection andretroviral-associated neurological disease). These treatments modulatethe immune system by inducing tolerance, orally or mucosally, e.g. byinhalation, using as tolerizers autoantigens, bystander antigens, ordisease-suppressive fragments or analogs of autoantigens or bystanderantigens. Such treatments are described, for example, in U.S. Pat. No.5,935,577 to Weiner et al. Autoantigens and bystander antigens aredefined below (for a general review of mucosal tolerance seeNagler-Anderson, C., Crit Rev Immunol 2000; 20(2):103-20). Intravenousadministration of autoantigens (and fragments thereof containingimmunodominant epitopic regions of their molecules) has been found toinduce immune suppression through a mechanism called clonal anergy.Clonal anergy causes deactivation of only immune attack T-cells specificto a particular antigen, the result being a significant reduction in theimmune response to this antigen. Thus, the autoimmune response-promotingT-cells specific to an autoantigen, once anergized, no longerproliferate in response to that antigen. This reduction in proliferationalso reduces the immune reactions responsible for autoimmune diseasesymptoms (such as neural tissue damage that is observed in MS). There isalso evidence that oral administration of autoantigens (orimmunodominant fragments) in a single dose and in substantially largeramounts than those that trigger “active suppression” may also inducetolerance through anergy (or clonal deletion).

A method of treatment has also been disclosed that proceeds by activesuppression. Active suppression functions via a different mechanism fromthat of clonal anergy. This method, discussed extensively in PCTApplication PCT/US93/01705, involves oral or mucosal administration ofantigens specific to the tissue under autoimmune attack. These arecalled “bystander antigens”. This treatment causes regulatory(suppressor) T-cells to be induced in the gut-associated lymphoid tissue(GALT), or bronchial associated lymphoid tissue (BALT), or mostgenerally, mucosa associated lymphoid tissue (MALT) (MALT includes GALTand BALT). These regulatory cells are released in the blood or lymphatictissue and then migrate to the organ or tissue afflicted by theautoimmune disease and suppress autoimmune attack of the afflicted organor tissue. The T-cells elicited by the bystander antigen (whichrecognize at least one antigenic determinant of the bystander antigenused to elicit them) are targeted to the locus of autoimmune attackwhere they mediate the local release of certain immunomodulatory factorsand cytokines, such as transforming growth factor beta (TGF-β),interleukin-4 (IL-4), and/or interleukin-10 (IL-10). Of these, TGF-β isan antigen-nonspecific immunosuppressive factor in that it suppressesimmune attack regardless of the antigen that triggers the attack.(However, because oral or mucosal tolerization with a bystander antigenonly causes the release of TGF-β in the vicinity of autoimmune attack,no systemic immunosuppression ensues.) IL-4 and IL-10 are alsoantigen-nonspecific immunoregulatory cytokines. IL-4 in particularenhances T helper type 2 (Th₂) response, i.e., acts on T-cell precursorsand causes them to differentiate preferentially into Th₂ cells at theexpense of Th₁ responses. IL-4 also indirectly inhibits Th₁exacerbation. IL-10 is a direct inhibitor of Th₁ responses. After orallytolerizing mammals afflicted with autoimmune disease conditions withbystander antigens, increased levels of TGF-β, IL-4 and IL-10 areobserved at the locus of autoimmune attack (Chen, Y. et al., Science,265:1237-1240, 1994). The bystander suppression mechanism has beenconfirmed by von Herreth et al., (J. Clin. Invest., 96:1324-1331,September 1996).

More recently, oral-mediated immunomodulation resulting in oraltolerance has been effectively applied in treatment of animal models ofinflammatory bowel disease by feeding probiotic bacteria (Dunne, C, etal., Antonie Van Leeuwenhoek 1999 July-November; 76(1-4):279-92),autoimmune glomerulonephritis by feeding glomerular basement membrane(Reynolds, J. et al., J Am Soc Nephrol 2001 January; 12(1): 61-70)experimental allergic encephalomyelitis (EAE, which is the equivalent ofmultiple sclerosis or MS), by feeding myelin basic protein (MBP),adjuvant arthritis and collagen arthritis, by feeding a subject withcollagen and HSP-65, respectively. A Boston based company calledAutoimmune has carried out several human experiments for preventingdiabetes, multiple sclerosis, rheumatoid arthritis and uveitis. Theresults of the human experiments have been less impressive than thenon-human ones, however there has been some success with the preventionof arthritis.

Immunomodulation by induction of oral tolerance to autoantigens found inatherosclerotic plaque lesions has also been investigated. Study of theepitopes recognized by T-cells and Ig titers in clinical andexperimental models of atherosclerosis indicated three candidateantigens for suppression of inflammation in atheromatous lesions:oxidized LDL, the stress-related heat shock protein HSP 65 and thecardiolipin binding protein beta 2GP1. U.S. patent application Ser. No.09/806,400 to Shoenfeld et al. (filed Sep. 30, 1999), which isincorporated herein in its entirety, discloses the reduction byapproximately 30% of atherogenesis in the arteries of geneticallysusceptible LDL-RD receptor deficient transgenic mice fed with oxidizedhuman LDL. This protective effect, however, was achieved by feeding acrude antigen preparation consisting of centrifuged, filtered andpurified human serum LDL which had been subjected to a lengthy oxidationprocess with Cu⁺⁺ or malondialdehyde (MDA). Although significantinhibition of atherogenesis was achieved, presumably via oral tolerance,no identification of specific lipid antigens or immunogenic LDLcomponents was made. Another obstacle encountered was the inherentinstability of the crude oxidized LDL in vivo, due to enzymatic activityand uptake of oxidized LDL by the liver and cellular immune mechanismsand its heterogeneity between different donors. It is plausible that astable, more carefully defined oxidized LDL analog would have providedimmunomodulation (e.g., by oral tolerance) of greater efficiency.

The induction of immune tolerance and subsequent prevention orinhibition of autoimmune inflammatory processes has been demonstratedusing exposure to suppressive antigens via mucosal sites other than thegut. The membranous tissue around the eyes, and the mucosa of the nasalcavity, as well as the gut, are exposed to many invading as well asself-antigens and possess mechanisms for immune reactivity. Thus, Rossi,et al. (Scand J Immunol 1999 August; 50(2):177-82) found that nasaladministration of gliadin was as effective as intravenous administrationin downregulating the immune response to the antigen in a mouse model ofceliac disease. Similarly, nasal exposure to acetylcholine receptorantigen was more effective than oral exposure in delaying and reducingmuscle weakness and specific lymphocyte proliferation in a mouse modelof myasthenia gravis (Shi, F D. et al., J Immunol 1999 May 15; 162 (10):5757-63). Therefore, immunogenic compounds intended for mucosal as wellas intravenous or intraperitoneal administration should optimally beadaptable to nasal and other membranous routes of administration.

Thus, there is clearly a need for novel, well defined, syntheticoxidized phospholipid derivatives and related substances exhibitingenhanced metabolic stability and efficient immunomodulation, induced by,e.g., oral, intravenous, intraperitoneal and mucosal administration.

Synthesis of Oxidized Phospholipids

Modification of phospholipids has been employed for a variety ofapplications. For example, phospholipids bearing lipid-soluble activecompounds may be incorporated into compositions for trans-dermal andtrans-membranal application (U.S. Pat. No. 5,985,292 to Fournerou etal.) and phospholipid derivatives can be incorporated into liposomes andbiovectors for drug delivery (see, for example, U.S. Pat. Nos. 6,261,597and 6,017,513 to Kurtz and Betbeder, et al., respectively, and U.S. Pat.No. 4,614,796). U.S. Pat. No. 5,660,855 discloses lipid constructs ofaminomannose derivatized cholesterol suitable for targeting smoothmuscle cells or tissue, formulated in liposomes. These formulations areaimed at reducing restenosis in arteries, using PTCA procedures. The useof liposomes for treating atherosclerosis has been further disclosed inWO 95/23592, to Hope and Rodrigueza, who teach pharmaceuticalcompositions of unilamellar liposomes that may contain phospholipids.The liposomes disclosed in WO 95/23592 are aimed at optimizingcholesterol efflux from atherosclerotic plaque and are typicallynon-oxidized phospholipids.

Modified phospholipid derivatives mimicking platelet activating factor(PAF) structure are known to be pharmaceutically active in variety ofdisorders and diseases, effecting such functions as vascularpermeability, blood pressure, heart function inhibition etc. It has beensuggested that one group of these derivatives may have anti cancerousactivity (U.S. Pat. No. 4,778,912 to Inoue at al.). U.S. Pat. No.4,329,302 teaches synthetic phosphoglycerides compounds—lysolechitinderivatives—that are usable in mediating platelet activation. While thecompounds disclosed in U.S. Pat. No. 4,329,302 are either 1-O-alkylether or 1-O-fatty acyl phosphoglycerides, it was found that small chainacylation of lysolechitin gave rise to compounds with plateletactivating behavior, as opposed to long-chain acylation, and that the1-O-alkyl ether are biologically superior to the corresponding 1-O-fattyacyl derivatives in mimicking PAF.

The structural effect of various phospholipids on the biologicalactivity thereof has also been investigated by Tokumura et al. (Journalof Pharmacology and Experimental Therapeutics. July 1981, Vol. 219,No. 1) and in U.S. Pat. No. 4,827,011 to Wissner et al., with respect tohypertension.

Another group of modified phospholipid ether derivatives has beendisclosed in CH Pat. No. 642,665 to Berchtold. These modifiedphospholipid ether derivatives were found useful in chromatographicseparation, but might have some physiological effect.

Oxidation of phospholipids occurs in vivo through the action of freeradicals and enzymatic reactions abundant in atheromatous plaque. Invitro, preparation of oxidized phospholipids usually involves simplechemical oxidation of a native LDL or LDL phospholipid component.Investigators studying the role of oxidized LDL have employed, forexample, ferrous ions and ascorbic acid (Itabe, H., et al., J. Biol.Chem. 1996; 271:33208-217) and copper sulfate (George, J. et al.,Atherosclerosis. 1998; 138:147-152; Ameli, S. et al., ArteriosclerosisThromb Vasc Biol 1996; 16:1074-79) to produce oxidized, or mildlyoxidized phospholipid molecules similar to those associated with plaquecomponents. Similarly prepared molecules have been shown to be identicalto auto-antigens associated with atherogenesis (Watson A. D. et al., J.Biol. Chem. 1997; 272:13597-607) and able to induce protectiveanti-atherogenic immune tolerance (U.S. patent application Ser. No.09/806,400 to Shoenfeld et al., filed Sep. 30, 1999) in mice. Likewise,Koike (U.S. Pat. No. 5,561,052) discloses a method of producing oxidizedlipids and phospholipids using copper sulfate and superoxide dismutaseto produce oxidized arachidonic or linoleic acids and oxidized LDL fordiagnostic use. Davies et al. (J. Biol. Chem. 2001, 276:16015) teach theuse of oxidized phospholipids as peroxisome proliferator-activatedreceptor agonists.

1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC, seeExample I for a 2-D structural description) and derivatives thereof suchas 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) arerepresentative examples of oxidized esterified phospholipids that havebeen studied with respect to atherogenesis (see, for example, Boullieret al., J. Biol. Chem. 2000, 275:9163; Subbanagounder et al.,Circulation Research, 1999, pp. 311). The effect of different structuralanalogs that belong to this class of oxidized phospholipids has alsobeen studied (see, for example, Subbanagounder et al., Arterioscler.Thromb. Nasc. Biol. 2000, pp. 2248; Leitinger et al., Proc. Nat. Ac.Sci. 1999, 96:12010).

However, in vivo applications employing oxidized phospholipids preparedas above have the disadvantage of susceptibility to recognition, bindingand metabolism of the active component in the body, making dosage andstability after administration an important consideration.

Furthermore, the oxidation techniques employed are non-specific,yielding a variety of oxidized products, necessitating either furtherpurification or use of impure antigenic compounds. This is of evengreater concern with native LDL, even if purified.

Thus, there is a widely recognized need for, and it would be highlyadvantageous to have, a novel, synthetic oxidized phospholipid, improvedmethods of synthesizing same and uses thereof as immunomodulators,devoid of the above limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided acompound having the general formula I:

wherein:

n is an integer of 1-6, whereas if n=1, Cn, Bn, Rn, R′n and Y areabsent;

each of B₁, B₂, . . . Bn−1 and Bn is independently selected from thegroup consisting of oxygen, sulfur, nitrogen, phosphor and silicon,whereby each of said nitrogen, phosphor and silicon is substituted by atleast one substituent selected from the group consisting of hydrogen,lone pair electrons, alkyl, halo, cycloalkyl, aryl, hydroxy,thiohydroxy, alkoxy, aryloxy, thioaryloxy, thioalkoxy and oxo;

each of A₁, A₂, . . . An−1 and An is independently selected from thegroup consisting of CR″R′″, C═O and C═S,

Y is selected from the group consisting of hydrogen, alkyl, aryl,cycloalkyl, carboxy, saccharide, phosphoric acid, phosphoryl choline,phosphoryl ethanolamine, phosphoryl serine, phosphoryl cardiolipin,phosphoryl inositol, ethylphosphocholine, phosphorylmethanol,phosphorylethanol, phosphorylpropanol, phosphorylbutanol,phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine and phsophoglycerol; and

each of X₁, X₂, . . . Xn−1 is independently a saturated or unsaturatedhydrocarbon having the general formula II:

wherein:

m is an integer of 1-26; and

Z is selected from the group consisting of:

whereas:

W is selected from the group consisting of oxygen, sulfur, nitrogen andphosphor, whereby each of said nitrogen and phosphor is substituted byat least one substituent selected from the group consisting of hydrogen,lone pair electrons, alkyl, halo, cycloalkyl, aryl, hydroxy,thiohydroxy, alkoxy, aryloxy, thioaryloxy, thioalkoxy and oxo; and

in at least one of X₁, X₂, . . . Xn−1 Z is not hydrogen; and wherein:

each of R₁, R′₁, R₂, . . . Rn−1, Rn, R′n, each of R″ and R′″ and each ofRa, R′a, Rb, R′b, . . . Rm−1, R′m−1, Rm and R′m is independentlyselected from the group consisting of hydrogen, a bond, alkyl, alkenyl,alkylnyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo,trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, phosphonate, phosphate, phosphinyl, sulfonyl, sulfinyl,sulfonamide, amide, carbonyl, thiocarbonyl, C-carboxy, O-carboxy,C-carbamate, N-carbamate, C-thiocarboxy, S-thiocarboxy and amino, or,alternatively, at least two of R1, R′₁, R2, . . . Rn−1, Rn and R′nand/or at least two of Ra, R′a, Rb, R′b, . . . Rm−1, R′m−1, Rm and R′mform at least one four-, five- or six-membered aromatic, heteroaromatic,alicyclic or heteroalicyclic ring; and

each of C₁, C₂, . . . , Cn−1, Cn, and each of Ca, Cb, . . . Cm−1 and Cmis a chiral or non-chiral carbon atom, whereby each chiral carbon atomhas a S-configuration and/or a R-configuration,

a pharmaceutically acceptable salt, a prodrug, a hydrate or a solvatethereof.

According to further features in preferred embodiments of the inventiondescribed below, at least one of A₁, A₂, . . . and An−1 is CR″R′″, andat least one of these A₁, A₂, . . . and An−1 is linked to a X₁, X₂ . . .or Xn−1 which comprises a Z different than hydrogen.

According to still further features in the described preferredembodiments n equals 3 and at least one of A₁ and A₂ is CR″R′″.Preferably, A₂ is CR″R′″ and X₂ comprises a Z different than hydrogen.Further preferably, each of A₁ and A₂ is CR″R′″.

According to still further features in the described preferredembodiments Z is selected from the group consisting of

whereby W is preferably oxygen and each of R″ and R′″ is independentlyselected from the group consisting of hydrogen and alkyl.

According to still further features in the described preferredembodiments n equals 1 and at least one of R₁ and R′₁ is a phosphate ora phosphonate.

According to still further features in the described preferredembodiments n equals 5 or 6 and at least one of R₁, R′₁ and at least oneof Rn and R′n form at least one heteroalicyclic ring, e.g., amonosaccharide ring.

According to another aspect of the present invention there is provided apharmaceutical composition comprising, as an active ingredient, thecompound described hereinabove and a pharmaceutically acceptablecarrier.

According to further features in preferred embodiments of the inventiondescribed below, the pharmaceutical composition is packaged in apackaging material and identified in print, in or on said packagingmaterial, for use in the treatment or prevention of an inflammationassociated with an endogenous oxidized lipid, as is detailedhereinbelow.

According to still further features in the described preferredembodiments, the pharmaceutical composition further comprises at leastone additional compound capable of treating or preventing theinflammation associated with an endogenous oxidized lipid, as isdetailed hereinbelow.

According to still another aspect of the present invention, there isprovided a method of treating or preventing an inflammation associatedwith an endogenous oxidized lipid, which comprises administering to asubject in need thereof a therapeutically effective amount of at leastone oxidized lipid, thereby treating or preventing the inflammationassociated with an endogenous oxidized lipid in the subject.

According to still further features in the described preferredembodiments the oxidized lipid is selected from the group consisting ofan oxidized phospholipid, a platelet activating factor, a plasmalogen, asubstituted or unsubstituted 3-30 carbon atoms hydrocarbon terminatingwith an oxidized group, an oxidized sphingolipids, an oxidizedglycolipid, an oxidized membrane lipid and any analog or derivativethereof.

According to still further features in the described preferredembodiments, the oxidized lipid has the general formula I depictedhereinabove.

According to still further features in the described preferredembodiments the oxidized lipid is selected from the group consisting of:1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC),1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC),1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphocholine,1-hexadecyl-2-acetoyl-sn-glycero-3-phosphocholine,1-octadecyl-2-acetoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-butyroyl-sn-glycero-3-phosphocholine,1-octadecyl-2-butyroyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine,1-octadecenyl-2-acetoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-(homogammalinolenoyl)-sn-glycero-3-phosphocholine,1-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-eicosapentaenoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine,1-octadecyl-2-methyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-butenoyl-sn-glycero-3-phosphocholine, Lyso PAF C16, LysoPAF C18,1-O-1′-(Z)-hexadecenyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine,1-O-1-(Z)-hexadecenyl-2-oleoyl-sn-glycero-3-phosphocholine,1-O-1-(Z)-hexadecenyl-2-arachidonoyl-sn-glycero-3-phosphocholine,1-O-1′-(Z)-hexadecenyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine,1-O-1-(Z)-hexadecenyl-2-oleoyl-sn-glycero-3-phosphoethanolamine,1-O-1′-(Z)-hexadecenyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine,and1-O-1′-(Z)-hexadecenyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine.

According to still further features in the described preferredembodiments, the method further comprises administering to the subject atherapeutically effective amount of at least one additional compoundcapable of treating or preventing an inflammation associated withendogenous oxidized LDL.

The at least one additional compound is preferably selected from thegroup consisting of a HMGCoA reductase inhibitor (a statin), a mucosaladjuvant, a corticosteroid, a steroidal anti-inflammatory drug, anon-steroidal anti-inflammatory drug, an analgesic, a growth factor, atoxin, a HSP, a Beta-2-glycoprotein I, a cholesteryl ester transferprotein (CETP) inhibitor, a perixosome proliferative activated receptor(PPAR) agonist, an anti-atherosclerosis drug, an anti-proliferativeagent, ezetimide, nicotinic acid, a squalen inhibitor, an ApoE Milano,and any derivative and analog thereof.

The inflammation according to the present invention is associated withdiseases and disorders such as, for example, idiopathic inflammatorydiseases or disorders, chronic inflammatory diseases or disorders, acuteinflammatory diseases or disorders, autoimmune diseases or disorders,infectious diseases or disorders, inflammatory malignant diseases ordisorders, inflammatory transplantation-related diseases or disorders,inflammatory degenerative diseases or disorders, diseases or disordersassociated with a hypersensitivity, inflammatory cardiovascular diseasesor disorders, inflammatory cerebrovascular diseases or disorders,peripheral vascular diseases or disorders, inflammatory glandulardiseases or disorders, inflammatory gastrointestinal diseases ordisorders, inflammatory cutaneous diseases or disorders, inflammatoryhepatic diseases or disorders, inflammatory neurological diseases ordisorders, inflammatory musculo-skeletal diseases or disorders,inflammatory renal diseases or disorders, inflammatory reproductivediseases or disorders, inflammatory systemic diseases or disorders,inflammatory connective tissue diseases or disorders, inflammatorytumors, necrosis, inflammatory implant-related diseases or disorders,inflammatory aging processes, immunodeficiency diseases or disorders,proliferative diseases and disorders and inflammatory pulmonary diseasesor disorders, as is detailed hereinbelow.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing novel synthetic oxidizedlipids, devoid of the limitations associated with the presently knownsynthetic oxidized lipids and methods of treating or preventing aninflammation associated with an endogenous oxidized lipid utilizingsynthetic oxidized lipids.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a flow chart depicting the synthesis of 2,5′ Aldehyde lechitinether, 1-hexadecyl-2-(5′-oxo-pentanyl)-sn-glycero-3-phosphocholine (forD-ALLE) or 3-hexadecyl-2-(5′-oxo-pentanyl)-sn-glycero-1-phosphocholine(for L-ALLE) (ALLE), according to the synthesis method of the presentinvention.

FIG. 2 is a flow chart depicting the synthesis of POVPC according to thepresent invention.

FIG. 3 is a graphic representation demonstrating inhibition of earlyatherogenesis in apoE-deficient mice by intra peritoneal immunizationwith mixed D- and L-isomers of ALLE. 5-7 week old Apo-E KO mice wereimmunized with 150 μg/mouse mixed D- or L-isomers of ALLE coupled topurified tuberculin protein derivative (ALLE L+D) (n=6), purifiedtuberculin protein derivative alone (PPD) (n=5) or unimmunized (CONTROL)(n=7). Atherogenesis is expressed as the area of atheromatous lesions inthe aortic sinus 4.5 weeks following the 4^(th) immunization.

FIG. 4 is a graphic representation demonstrating inhibition of earlyatherogenesis in Apo-E KO mice by oral administration of ALLE. 6-7.5week old Apo-E KO mice were fed mixed D- and L-isomers of ALLE: 10μg/mouse (ALLE L+D 10 μg) (n=11) or 1 mg/mouse (ALLE L+D 1 mg) (n=11);or PBS (CONTROL) (n=12) every other day for 5 days. Atherogenesis isexpressed as the area of atheromatous lesions in the aortic sinus 8weeks after the last feeding.

FIG. 5 is a graphic representation demonstrating inhibition of earlyatherogenesis in Apo-E KO mice by oral and nasal administration ofL-ALLE. 7-10 week old Apo-E KO mice were either fed 1 mg/mouse L-ALLEevery other day for 5 days (OT L-ALLE) (n=11) or intranasallyadministered with 10 μg/mouse L-ALLE every other day for 3 days (NTL-ALLE) (n=11). Control mice were fed an identical volume (0.2 ml) ofPBS (PBS ORAL) (n=12). Atherogenesis is expressed as the area ofatheromatous lesions in the aortic sinus 8 weeks after the last oral ornasal exposure.

FIG. 6 is a graphic representation demonstrating suppression of immunereactivity to atherosclerotic plaque antigens induced by oral feedingwith the synthetic oxidized phospholipids L-ALLE and POVPC. 6 week oldmale Apo-E KO mice were fed either 1 mg/mouse L-ALLE (L-ALLE) (n=2) orPOVPC (POVPC) (n=3) in 0.2 ml PBS; or PBS alone (CONTROL) (n=3) everyother day for 5 days. One week following the last feeding the mice wereimmunized with a single subcutaneous injection of 50 μg Human oxidizedLDL antigen. 7 days later T-cells from inguinal lymph node were preparedas described in Materials and Methods section that follows, and exposedto the sensitizing Human ox-LDL antigen for in-vitro assessment ofproliferation. Proliferation, indicating immune reactivity, is expressedas the ratio between incorporation of labeled thymidine into theT-cell's DNA in the presence and absence of human ox-LDL antigen(stimulation index, S.I.).

FIG. 7 is a graphic representation demonstrating inhibition ofprogression of late-stage atherogenesis in Apo-E KO mice by oraladministration of the synthetic oxidized phospholipids D-ALLE, L-ALLE orPOVPC. 24.5 week old Apo-E KO mice were fed 1 mg/mouse L-ALLE (L-ALLE)(n=11), D-ALLE (D-ALLE) (n=9) or POVPC (POVPC) (n=10) every other dayfor 5 days, at 4 week intervals over a 12 week period. Control mice werefed an identical volume (0.2 ml) and regimen of PBS (CONTROL) (n=10).Atherogenesis is expressed as the area of atheromatous lesions in theaortic sinus 12 weeks after the first feeding, as compared to the lesionscores of untreated 24.5 week old mice before feeding (sacrificed atTime 0).

FIG. 8 is a graphic representation demonstrating reduction oftriglyceride content of VLDL in Apo-E KO mice induced by feedingsynthetic oxidized phospholipids D-ALLE, L-ALLE or POVPC. 24.5 weeks oldApo-E KO mice were fed 1 mg/mouse L-ALLE (triangle) (n=11), D-ALLE(inverted triangle) (n=9) or POVPC (square) (n=10) every other day for 5days, at 4 weeks intervals over a 12 weeks period. Control mice were fedan identical volume (0.2 ml) and regimen of PBS (circle) (n=10).Triglyceride content (Tg, mg/ml) was measured 9 weeks from t=0, byenzymatic colorimetric method in the VLDL fractions following separationof pooled blood samples on FPLC, as described in the materials andmethods section that follows.

FIG. 9 is a graphic representation demonstrating reduction ofcholesterol content of VLDL in Apo-E KO mice induced by feedingsynthetic oxidized phospholipids D-ALLE, L-ALLE or POVPC. 24.5 weeks oldApo-E KO mice were fed 1 mg/mouse L-ALLE (triangle) (n=11), D-ALLE(inverted triangle) (n=9) or POVPC (square) (n=10) every other day for 5days, at 4 weeks intervals over a 12 weeks period. Control mice were fedan identical volume (0.2 ml) and regimen of PBS (circle) (n=10).Cholesterol content (Cholesterol, mg/ml) was measured 9 weeks from t=0,by enzymatic colorimetric method in the VLDL fractions followingseparation of pooled blood samples on FPLC, as described in thematerials and methods section that follows.

FIG. 10 presents 2D structural descriptions of1-Hexadecyl-2-(5′-Carboxy-butyl)-sn-glycero-3-phosphocholine (CI-201,Compound VII),1-Hexadecyl-2-(5′,5′-Dimethoxy-pentyloxy)-sn-glycero-3-phosphocholine(Compound VIIIa) and1-Hexadecyl-2-(5′,5′-Diethoxy-pentyloxy)-sn-glycero-3-phosphocholine(Compound VIIIb).

FIG. 11 is a graphic representation demonstrating inhibition of earlyatherogenesis in Apo-E KO mice by oral administration of CI-201. 12 weekold Apo-E KO mice were fed CI-201: 0.025 mg/mouse (n=14); or 0.2 ml PBS(CONTROL) (n=15) every day for 8 weeks (5 times a week). Atherosclerosisis expressed as the area of atheromatous lesion in the aortic sinus 11weeks after the first feeding.

FIGS. 12 a-d present photographs demonstrating the cytokine expressionlevels in the aorta of mice treated with ALLE, CI-201, its ethyl acetalderivative (Et-acetal), its methyl acetal derivative (Me-acetal), oxLDLor PBS. Particularly, FIGS. 12 a and 12 b present the elevation of IL-10expression level in the aorta of mice treated with ALLE, CI-201,Et-acetal, Me-acetal and oxLDL as compared with non-treated mice (PBS)and the reduced IFN-gamma expression levels in aortas from mice treatedwith ALLE, CI-201, Me-acetal and oxLDL as compared with PBS treatedmice, and FIGS. 12 c and 12 d present the reduced IL-12 expression inmice treated with ALLE, CI-201 and Et-acetal as compared with PBStreated group. 10-12 weeks old Apo-E KO mice were fed 1 mg/mouse/0.2 mlof the tested antigen (ALLE, CI-201, Et-acetal, Me-acetal) or 0.1mg/mouse/0.2 ml oxLDL or administered with 0.2 ml PBS. Oraladministrations took place 5 times every other day and the cytokineexpression was evaluated 8 weeks after the last oral administration.

FIG. 13 presents a bar graph demonstrating the attenuation ofatherogenesis in LDL-RD mice by oral administration of oxLDL. LDL-RDmice were fed with PBS, or 10, 100 and 1,000 μg/dose oxLDL, 5 timesevery other day. Atherogenesis is expressed as the area of atheromatouslesions in the aortic sinus, 5 weeks after the last feeding.

FIGS. 14 a-b present bar graphs demonstrating the inhibition ofatherogenesis in Apo-E KO mice by oral administration of CI-201. Apo-EKO mice were fed with PBS (control) or 0.1, 1 and 10 μg/dose CI-201, atthree sets at the beginning of each month, 5 times every other day ineach set. Atherogenesis is expressed as the area of atheromatous lesionsin the aortic sinus, 12 weeks after the first feeding. FIG. 14 apresents the extent of atherosclerosis in each group. FIG. 14 b presentsthe dramatic effect of the low dose CI-201 treatment on atherosclerosis,as compared with the “base line” group (sacrificed at day 0) and thecontrol group.

FIGS. 15 a-b present bar graphs demonstrating the elevation in serumlevels of IL-10 (FIG. 15 a) and the prevention of SAA elevation (FIG. 15b) in Apo-E KO mice treated by CI-201. Apo-E KO mice were fed with PBS(control) or CI-201, 5 times every other day. Serum was collected at thebeginning of the experiment, 2 weeks and 4 weeks after the firstfeeding. Markers levels were evaluated as described in the Materials andMethods section that follows.

FIGS. 16 a-b present photographs (FIG. 16 a) and a graphicrepresentation (FIG. 16 b) demonstrating the cytokine expression levelsin the aorta of mice treated with CI-201 or PBS. Particularly, FIGS. 16a and 16 b present the elevation of IL-10 expression level in the aortaof mice treated with CI-201, as compared with non-treated mice (PBS) andthe reduced IFN-gamma expression levels in aortas from mice treated withCI-201, as compared with PBS treated mice. Apo-E KO mice were fed with 1mg/mouse CI-201 or with 0.2 ml/mouse PBS, 5 times every other day. Theexpression of the anti-inflammatory cytokine IL-10 and thepro-inflammatory cytokine IFN-γ were determined 8 weeks after the lastfeeding.

FIG. 17 presents photographs demonstrating aorta-targeted CI-201 oraltreatment in Apo-E KO mice. While in the aorta CI-201 treatment inducedelevation of IL-10 expression level and reduction of IFN-gammaexpression levels, as compared with the PBS treatment, no differenceswere observed in cytokine expression in the spleen and in the smallintestine between the CI-201 treated group and the control, PBS treated,group.

FIG. 18 is a graphic presentation of the study design for evaluating theattenuation of Adjuvant-induced arthritis (AIA) in rats pre-treated withCI-201.

FIG. 19 presents a bar graph demonstrating the effect of oraladministration of CI-201 in Adjuvant induced arthritis (AIA)-inducedrats in terms of paw swelling. Lewis rats were fed with CI-201 or PBS(CONTROL), 5 times every other day, and where thereafter injectedintradermally with a tuberculosis suspension.

FIG. 20 is a graphic presentation of the study design for evaluating theattenuation of Adjuvant-induced arthritis (AIA) in rats continuouslytreated with CI-201.

FIG. 21 presents a bar graph demonstrating the effect of oraladministration of CI-201 in AIA-induced Lewis rats in terms of pawswelling. Lewis rats were fed with CI-201 or PBS (CONTROL), 5 timesevery other day, subjected thereafter to arthritis induction and werethen continuously treated with CI-201 by feeding 3 times a week.

FIG. 22 presents comparative plots demonstrating the arthritis scoreassessment monitored during arthritis development in rats treated withvarious concentrations of CI-201, as compared with PBS-treated rats.

FIG. 23 presents comparative plots demonstrating the percentage of ratshaving arthritis symptoms following treatment with PBS (control) andvarious concentrations of CI-201.

FIG. 24 presents a bar graph demonstrating the effect on earlyatherogenesis in Apo-E KO mice induced by oral administration of thepre-oxidized Compound V. 8-10 week old female Apo-E KO mice were fedwith Compound V: 5 mg/mouse (n=6), 1 mg/mouse (n=6), 0.2 mg/mouse (n=6)or PBS (control) (n=7) every other day for 5 days. Atherogenesis isexpressed as the area of atheromatous lesions in the aortic sinus 8weeks after the last feeding.

FIG. 25 presents bar graphs demonstrating the effect on atherogenesis inApo-E KO mice induced by oral administration of the pre-oxidizedCompound V. 23-26 week old Apo-E KO mice were either sacrificed at thebeginning of experiment (baseline B. L. group, n=10) or fed with PBS(control, n=11) or 0.1 μg/dose Compound V (n=10), at three sets at thebeginning of each month, 5 times every other day in each set.Atherogenesis is expressed as the area of atheromatous lesions in theaortic sinus, 12 weeks after the first feeding.

FIGS. 26 a-b are scatter plots and graphs illustrating that in vivoadministration of CI-201 reduces IFN-γ production by T cells. (FIG. 26a) C57BL/6 mice were fed in alternate days from day −10 to day +10 withCI-201 (0.1 μg/feed) of PBS. On day 0, mice were immunized withovalbumin in CFA. On day 10, draining lymph nodes were excised and cellsstimulated with ovalbumin. Cells were expanded for a week and thenre-stimulated for 4 hours with plate bound anti-CD3 and anti-CD28 (5μg/ml) in the presence of monensin (2 μM) and tested for cytokineexpression by intracellular staining. Results are gated on CD4+ T cells.(FIG. 26 b) SJL mice were orally administered with CI-201 (10 and 100μg/mouse/feed) or PBS, 5 times once a day, every other day for a totalof 5 doses and then immunized with PLP peptide 139-151 in CFA followedby 4 more administrations every other day. Lymph nodes were collected 10days later, stimulated ex vivo with 10 μg/ml of PLP 139-151 peptide or0.5 μg/ml of anti CD3 and three days later supernatants were collectedand tested by ELISA for cytokine production.

FIG. 27 is a bar graph illustrating the preferential binding of³H—CI-201 to professional antigen presenting cells. ³H—CI-201 uptake wasevaluated at the indicated time points in monocytes, dendritic, T and Bcell lines. Chinese hamster ovary cells (CHO) served as a control cellline.

FIGS. 28 a-n are bar graphs illustrating that CI-201 inhibits p40 butnot the production of other pro inflammatory cytokines by activatedBMDC's. BMDC's were incubated for 1 hour with various CI-201concentrations prior to activation with the indicated agonists at theconcentrations listed in the materials and methods. Supernatant werecollected 24 hours following activation and the levels of p40 (FIGS. 28a-f) and TNF-α (FIGS. 28 g-l) were measured by ELISA.

BMDC's were incubated for 1 hour with different CI-201 concentrationsprior to activation with 10 μg/ml of PGN. Supernatant were collected 24hours following activation and the levels of IL-6 (FIG. 3M) and IL-1(FIG. 3N) were measured.

FIGS. 29 a-d are bar graphs illustrating that CI-201 alters p40 and p19a RNA expression level. Purified BM CD11c+ DC's were incubated for 1hour with CI-201 (20 μg/ml) prior to activation with PGN (10 μg/ml) andRNA was isolated at the indicated time points. Q-PCR was performed forIL-12/IL-23p40 (FIG. 29 a), IL-12p35 (FIG. 29 b), IL-23p19 (FIG. 29 c)and IL-27p28 (FIG. 29 d) All results were normalized to GAPDH.

FIGS. 30 a-b are bar graphs illustrating that CI-201 inhibits p40 butnot TNF-α production by human DC's. Mo-Dc's were incubated for 1 hourwith various CI-201 concentrations prior to activation with 10 μg/ml ofPGN. Supernatants were collected 24 hours following activation and thelevels of p40 (FIG. 30 a) and TNF-α (FIG. 30 b) were measured by ELISA.

FIGS. 31 a-d are bar graphs illustrating that CI-201 effects p40 andTNF-α production by PBMC's. PBMC's were isolated from two separatedonors and incubated for 1 hour with various CI-201 concentrations priorto activation with 10 μg/ml of PGN. Supernatant were collected 24 hoursfollowing activation and the levels of p40 (FIGS. 31 a-b) and TNF-α(FIG. 31 c-d) were measured by ELISA.

FIGS. 32 a-b are bar graphs illustrating that CI-201 does not impair p40production by Monocytes/Macrophages. Purified splenic CD11b monocytesand macrophages were resistant to incubated for 1 hour with variousCI-201 concentrations prior to activation with 10 μg/ml of PGN.Supernatants were collected 24 hours following activation and the levelsof p40 (FIG. 32 a) and TNF-α (FIG. 32 b) were measured by ELISA.

FIGS. 33 a-m are structure-function analysis with CI-201 derivatives.BMDC's were incubated for 1 h with various CI-201 concentrations (FIG.33 a) and either newly synthesized or commercially available CI-201analogous (FIGS. 33 b-m) prior to activation with 10 μg/ml of PGN.Supernatant were collected 24 hours following activation and the levelsof p40 was measured by ELISA.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods and compositions employing oxidizedlipids, which can be utilized in treating or preventing an inflammationassociated with endogenous oxidized lipids. Particularly, the presentinvention is of (i) novel oxidized lipids; (ii) pharmaceuticalcompositions containing same; (iii) methods employing the novel oxidizedlipids, as well as other oxidized lipids, for treating or preventing aninflammation associated with endogenous oxidized lipids, and therebytreating or preventing inflammation-associated diseases and disorderssuch as, but not limited to, atherosclerosis, cardiovascular diseases,cerebrovascular diseases, peripheral vascular diseases, stenosis,restenosis, in-stent-stenosis, autoimmune diseases or disorders,inflammatory diseases or disorders, infectious diseases or disorders andproliferative disease or disorders.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Experimental and clinical evidence indicates a causative role foroxidized LDL (ox LDL) and LDL components in the etiology of an excessiveinflammatory response in atherosclerosis. Both cellular and humoralimmune reactivity to plaque related oxidized LDL have been demonstrated,suggesting an important anti-oxidized LDL autoimmune component inatherogenesis. Thus, LDL, oxidized LDL and components thereof, have beenthe targets of numerous therapies for prevention and treatment of heartdisease, cerebral-vascular disease and peripheral vascular disease.

Prior art studies associated with the role of oxidized LDL andcomponents thereof in reducing the immune response to endogenous (e.g.,plaque related) oxidized LDL employed either a crude antigen preparationconsisting of centrifuged, filtered and purified human serum LDL whichhad been subjected to a lengthy oxidation process with Cu⁺⁺ or MDA, orsynthetically prepared oxidized LDL analogs. Since phospholipids areconsidered as active LDL components, studies with synthetically preparedoxidized LDL analogs typically involved oxidized phospholipids (e.g.,POVPC and PGPC).

Although the prior art teaches that oral administration of oxidized LDLcan result in 30% reduction in atherogenesis, thus suggesting aprotective effect of oxidized LDL, presumably via oral tolerance, noidentification of specific lipid antigens or immunogenic LDL componentswas made. Another obstacle encountered by these prior art studies wasthe inherent instability of the crude oxidized LDL in vivo, due toenzymatic activity and uptake of oxidized LDL by the liver and cellularimmune mechanisms. Such an inherent instability is also associated within vivo applications that utilize synthetic oxidized LDL derivativessuch as POVPC and PGPC (described hereinabove).

Hence, hitherto, no direct correlation between exogenous oxidized LDL orcomponents thereof and endogenous oxidized LDL, in terms ofimmunomodulation, has been established. Oxidized LDL analogs, devoid ofthe inherent instability and other limitations involved with theadministration of oxidized LDL, which can modulate the immune and/orinflammatory response associated with endogenous oxidized LDL and otherendogenous oxidized lipids, have not been uncovered so far as well.

While conceiving the present invention, it was hypothesized thatsynthetically defined oxidized lipids in general and oxidized LDLanalogs in particular could modulate the immune reactivity to endogenousoxidized lipids in general and oxidized LDL in particular and thus beused in the treatment or prevention of a myriad of diseases anddisorders, associated with inflammation and/or altered immune response,such as, for example, atherosclerosis and related diseases or disorders,as well as other diseases and disorder associated with endogenousoxidized lipid.

Inflammation involved in atherogenesis often leads to complications suchas plaque rupture and thrombosis (Libby et al., Inflammation andatherosclerosis. Circulation. 2002; 105:1135-1143).

The presence of activated T lymphocytes in human atherosclerotic lesionmay imply their involvement in the disease initiation and progression(Ross R. Atherosclerosis—an inflammatory disease. NEJM. 1999;340:115-126). The major class of T lymphocytes, CD4+, can differentiateinto the lineages Th1 or Th2, which are functionally defined by theproduced cytokine: interferon (IFN)-γ, secreted from the Th1 cells, andinterleukin (IL)-4 secreted from the Th2 cells. Among the principleinducers of the Th1 and Th2 cells are IL-12 and IL-10, respectively(Daugherty A and Rateri DL, T lymphocytes in atherosclerosis theYin-Yang of Th1 and Th2 Influence on lesion formation, Circ Res. 2002;90:1039-1040; Hansson G K, Vaccination against atherosclerosis scienceor fiction. Circulation. 2002; 106:1599-1601).

T-lymphocytes isolated from whole blood in patients with acute coronarysyndromes or harvested from human carotid plaques have been shown tospecifically recognize Ox LDL and proliferate when exposed to Ox LDL(Hansson GK. Immune mechanisms in atherosclerosis. Arterioscler ThrombVasc Biol 2001; 21:1,876-90). Ox LDL and oxidized lipid byproductsthereof (e.g., oxidized phospholipids) are present withinatherosclerotic plaques (Witztum 2001, supra).

Hence, while oxidative modification of LDL can be a prerequisite forrapid accumulation of LDL in macrophages and foam cell formation, it canfurther induce immunogenic epitopes in the LDL molecule, which lead toformation of antibodies against Ox LDL.

Oxidized LDL epitopes therefore serve as important ligands, mediatingthe binding and clearance of oxidatively damaged lipoprotein particlesand apoptotic cells, and inducing an innate immune response whicheffects their removal. On the other hand the oxidized LDL epitopes canplay a role in the immune activation that characterizes the progressiveatherosclerotic plaque.

In view of the above, the present inventors postulated that compoundswhich can serve as oxidized LDL epitopes may modulate the immuneresponse, so as to induce a beneficial rather than deleterious effect onatherogenesis. In other words, it was postulated that administering,preferably orally, oxidized LDL analogs, such as oxidized phospholipids,would induce tolerance to the endogenous oxidized LDL formed duringatherogenesis and would thus reduce the inflammatory response theretoand attenuate atherogenesis progression.

Evidence supporting immunomodulation as a new therapeutic approach totreat atherosclerosis has recently been published (Nicoletti et al.Induction of neonatal tolerance to oxidized lipoprotein reducesatherosclerosis in Apo E knockout (Apo-E KO) mice. Mol. Med. 2000;6(4):283-290). It was shown that intraperitoneal injection of oxidizedLDL to (Apo-E KO) mice at birth induced T-cell tolerance due to clonaldeletion, reduced the immune response to oxidized LDL and, as a result,reduced susceptibility to atherosclerosis.

Adaptive and innate immunity have been implicated in the pathogenesis ofatherosclerosis as well as in many other disease and disorders. Giventheir abundance in the lesion, lipids are possible targets of theatherosclerosis-associated immune response. Recently it has been shownthat natural killer T (NKT) cells can recognize lipid antigens presentedby CD1 molecules. CD1 molecules present lipid antigens to T-cells,unlike the evolutionarily-related major histocompatibility complex (MHC)class I and II molecules, which display peptide antigens. Like MHC classII molecules, however, CD1 molecules consist of a heavy chain associatedwith the β₂-microglobulin (β₂M) light chain. Crystal structures of twoCD1 isoforms, human CD1b and mouse CD1d, show an overall domainorganization that resembles MHC class I molecules. Notably, theantigen-binding site in CD1 is hydrophobic, forming channels (CD1b) orpockets (mouse CD1d) that can accommodate hydrocarbon chains of lipids.A narrow opening between the α-helices permits the display of polarmoieties of the lipid in a region accessible for recognition by T-cellreceptors (TCRs). This system facilitates the binding of different lipidmolecules linked to diverse polar head groups, thereby creating anenormous pool of potential CD1-presented antigens (Zeng et al. Crystalstructure of mouse CD1: an MHC-like fold with a large hydrophobicbinding groove. Science 1997; 277:339-345).

CD1 molecules bind foreign lipid antigens as they survey the endosomalcompartments of infected antigen-presenting cells. Unlike T-cells thatrecognize CD1-restricted foreign lipids, CD1-restricted T cells that areself-antigen reactive, function as ‘auto-effectors’ that are rapidlystimulated to carry out helper and effector functions upon interactionwith CD1-expressing antigen-presenting cells. The functionaldistinctions between subsets of CD 1-restricted T-cells and the pathwaysby which these cells both influence the inflammatory and tolerogeniceffects of dendritic cells and activate natural killer cells and otherlymphocytes provide insight into how CD1-restricted T cells regulateantimicrobial responses, antitumor immunity and the balance betweentolerance and autoimmunity (Vincent et al. Understanding the function ofCD1-restricted T cells. Nat. Immunol. 2003; 4:517-23).

Tupin et al. (CD1d-dependent Activation of NKT Cells AggravatesAtherosclerosis. J Exp Med. 2004; 199:417-22) have explored the role ofCD1d-restricted NKT cells in atherosclerosis by using apolipoproteinE-deficient (apoE(−/−)) mice, and ApoE(−/−) mice crossed with CD1d(−/−)(CD1d(−/−)apoE(−/−)) mice that exhibited a 25% decrease in lesion sizecompared with apoE(−/−) mice. Administration ofalpha-galactosylceramide, a synthetic glycolipid that activates NKTcells via CD1d, induced a 50% increase in lesion size in apoE(−/−) mice,whereas it did not affect lesion size in apoE(−/−)CD1d(−/−) mice. Theseresults show that activation of CD1d-restricted NKT cells exacerbatesatherosclerosis. Zhou et al. (Editing of CD1d-bound lipid antigens byendosomal lipid transfer proteins. Science. 2004; 303:523-7) havereported that mice deficient in prosaposin, the precursor to a family ofendosomal lipid transfer proteins (LTP), exhibit specific defects inCD1d-mediated antigen presentation and lack Vα14 NKT cells. In vitro,saposins extracted monomeric lipids from membranes and from CD1, therebypromoting the loading as well as the editing of lipids on CD1. Transientcomplexes between CD1, lipid, and LTP suggested a “tug-of-war” model inwhich lipid exchange between CD1 and LTP is on the basis of theirrespective affinities for lipids. LTPs constitute a previously unknownlink between lipid metabolism and immunity and are likely to exert aprofound influence on the repertoire of self, tumor, and microbial lipidantigens.

Type-2 activation of macrophages (M2) is an alternative pathway to theclassic macrophage activation. These M2 cells are APC's that arepresented in the Lamina-Propria of the gut as part of the gut associatedimmune system. These M2 cells will response with IL-10 expressioninstead of the classic Th1 cytokine response of macrophages as describebelow.

Activated macrophages are used as antigen presenting cells (APCs).Antigen recognition by T cells is the key event controlling the adaptiveimmune response.

The classical pathway of IFN-γ dependent activation of macrophages byTh1-type responses is a well-established feature of cellular immunity.Macrophage activation depends on the products of specifically activatedT helper—Th1-type lymphocytes and natural killer cells—in particular,IFN-γ and cytokine network involving IL-12 and IL-18, which are producedby APCs. The concept of an alternative pathway of macrophage activationby the Th2-type cytokines IL-4 and IL-13, together with IL-10, hasgained credence in the past decade, to account for a distinctivemacrophages phenotype that is consistent with a different role inhumoral immunity and repair.

IL-4 and IL-13 up-regulates expression of the mannose receptor and MHCclass II molecules by macrophages, which stimulate endocytosis andantigen presentation.

Immunoglobulins and immune complexes can bind both activating andinhibitory receptors for Fc and for complement. Also, Fc-receptorligation induces marked effects on the release of cytokines, such asIL-12/IL-10 and IL-4, by APCs themselves and by other cells of theinnate and acquired immune systems (Gordon S. Alternative Activation ofMacrophages. Nat. Rev. Immunol. 3: 23-34; 2003).

Macrophages challenged with inflammatory stimuli (IFN-γ for example) andintroduced to immune complexes dramatically opposed in their action,instead of a Th1 response: elevated levels of IL-12 and moderate levelsof IL-10 there is a dramatic decrease in IL-12 and an increase in IL-10levels. IL-10 exerts immune-suppressive effects on macrophages.(Anderson, C. F. and Mosser, D. M. A Novel Phenotype for an ActivatedMacrophages: the Type 2 Activated Macrophage. J. Leukoc. Biol. 72:101-106; 2002). IL-10 acts on a distinct plasma-membrane receptor tothose for IL-4 and IL-13, and its effect on macrophage gene expressionare different, involving a more profound inhibition of a range ofantigen-presenting and effector functions, together with the activationof selected genes or functions (Gordon S. Alternative Activation ofMacrophages. Nat. Rev. Immunol. 3: 23-34; 2003).

Hence, in addition to its effect on atherosclerosis and other diseaseswhich are directly associated with oxidized LDL, it was furtherpostulated that the immunomodulation and the anti-inflammatory effectinduced by oxidized LDL (synthetic) analogs can be utilized in thetreatment and prevention of other disease and disorders, directly orindirectly associated with endogenous oxidized LDL and other oxidizedlipids. This was supported by several studies which were directed atimmunotherapy of human autoimmune disease such as rheumatoid arthritis(RA), type I diabetes, and multiple sclerosis, either by modulation ofindividual immune pathways involved in inflammation or by tolerizationto various antigens (Bielekova et al. Encephalitogenic potential of themyelin basic protein peptide (amino acids 83-99) in multiple sclerosis:Results of a phase II clinical trial with an altered peptide ligand.Nat. Med. 2000; 6:1167-1175; Kappos et al. Induction of anon-encephalitogenic type 2 T helper-cell autoimmune response inmultiple sclerosis after administration of an altered peptide ligand ina placebo-controlled, randomized phase II trial. Nat. Med. 2000;6:1176-1182).

Hence, there are ample data supporting the relation between lipids,inflammation and the immune system, indicating a direct linkagetherebetween.

In an attempt to improve treatment of inflammation and diseases anddisorders associated with oxidized lipids, the present inventors havedesigned novel synthetic oxidized phospholipids and structurally relatedcompounds, which are devoid of the limitations associated oxidized LDLand other known oxidized phospholipids and lipids (as delineatedhereinabove).

As is demonstrated in the Examples section that follows, while reducingthe present invention to practice, it was indeed confirmed that oraland/or mucosal administration of the newly designed oxidized LDL analogsmodulate the immune and/or inflammatory response to endogenous oxidizedLDL, thereby reducing the inflammatory response in inflammatory diseasessuch as atherosclerosis and rheumatoid arthritis. These results clearlydemonstrate the effect of exogenous oxidized lipids on inflammatory andimmune processes which involve endogenous oxidized lipids.

Thus, according to one aspect of the present invention there areprovided novel compounds, designed so as to mimic the immunomodulationeffect induced by oxidized LDL and/or an inflammation associated withoxidized LDL and/or other oxidized lipids, while avoiding thelimitations associated with oxidized LDL and other oxidized lipids andare thus highly suitable for oral/mucosal treatment of inflammatoryassociated diseases and disorders which involve oxidized lipids.

Since oxidized phospholipids are known as active components of ox LDLand further since biological membranes typically include phospholipids,and mainly phosphoglycerides, the compounds according to the presentinvention are structurally based on oxidized phospholipids in generaland oxidized phosphoglycerides in particular.

Each of the compounds according to the present invention has the generalformula I:

wherein:

n is an integer of 1-6, whereas if n=1, Cn, Bn, Rn, R′n and Y areabsent;

each of B₁, B₂, . . . Bn−1 and Bn is independently selected from thegroup consisting of oxygen, sulfur, nitrogen, phosphor and silicon,whereby each of the nitrogen, phosphor and silicon is substituted by atleast one substituent selected from the group consisting of hydrogen,lone pair electrons, alkyl, halo, cycloalkyl, aryl, hydroxy,thiohydroxy, alkoxy, aryloxy, thioaryloxy, thioalkoxy and oxo;

each of A₁, A₂, . . . An−1 and An is independently selected from thegroup consisting of CR″R′″, C═O and C═S,

Y is selected from the group consisting of hydrogen, alkyl, aryl,cycloalkyl, carboxy, saccharide, phosphoric acid, phosphoryl choline,phosphoryl ethanolamine, phosphoryl serine, phosphoryl cardiolipin,phosphoryl inositol, ethylphosphocholine, phosphorylmethanol,phosphorylethanol, phosphorylpropanol, phosphorylbutanol,phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine and phsophoglycerol; and

each of X₁, X₂, . . . Xn−1 is independently a saturated or unsaturatedhydrocarbon having the general formula II:

wherein:

m is an integer of 1-26; and

Z is selected from the group consisting of:

whereas:

W is selected from the group consisting of oxygen, sulfur, nitrogen andphosphor, whereby each of the nitrogen and phosphor is substituted by atleast one substituent selected from the group consisting of hydrogen,lone pair electrons, alkyl, halo, cycloalkyl, aryl, hydroxy,thiohydroxy, alkoxy, aryloxy, thioaryloxy, thioalkoxy and oxo; and

in at least one of X₁, X₂, . . . Xn−1 Z is not hydrogen; and wherein:

each of R₁, R′₁, R₂, . . . Rn−1, Rn, R′n, each of R″ and R′″ and each ofRa, R′a, Rb, R′b, . . . Rm−1, R′m−1, Rm and R′m is independentlyselected from the group consisting of hydrogen, a bond, alkyl, alkenyl,alkylnyl, cycloalkyl, aryl, heteroaryl, halo, trihalomethyl, hydroxy,alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, phosphonate,phosphate, phosphinyl, sulfonyl, sulfinyl, sulfonamide, amide, carbonyl,thiocarbonyl, C-carboxy, O-carboxy, C-carbamate, N-carbamate,C-thiocarboxy, S-thiocarboxy and amino, or, alternatively, at least twoof R₁, R′₁, R2, . . . Rn−1, Rn and R′n and/or at least two of Ra, R′a,Rb, R′b, . . . Rm−1, R′m−1, Rm and R′m form at least one four-, five- orsix-membered aromatic, heteroaromatic, alicyclic or heteroalicyclicring; and

each of C₁, C₂, . . . , Cn−1, Cn, and each of Ca, Cb, . . . Cm−1 and Cmis a chiral or non-chiral carbon atom, whereby each chiral carbon atom,has a S-configuration and/or a R-configuration,

a pharmaceutically acceptable salt, a prodrug, a hydrate or a solvatethereof.

It will be appreciated by one of ordinary skill in the art that thefeasibility of each of the substituents (e.g., R₁-Rn, Ra-Rm, R″, R′″) tobe located at the indicated positions depends on the valency andchemical compatibility of the substituent, the substituted position andother substituents. Hence, the present invention is aimed atencompassing all the feasible substituents for any position.

As used herein throughout, the term “alkyl” refers to a saturatedaliphatic hydrocarbon including straight chain and branched chaingroups. Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever anumerical range; e.g., “1-20”, is stated herein, it implies that thegroup, in this case the alkyl group, may contain 1 carbon atom, 2 carbonatoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. Morepreferably, the alkyl is a medium size alkyl having 1 to 10 carbonatoms. Most preferably, unless otherwise indicated, the alkyl is a loweralkyl having 1 to 4 carbon atoms. The alkyl group may be substituted orunsubstituted. When substituted, the substituent group can be, forexample, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide,sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, oxo, carbonyl,thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, andamino, as these terms are defined herein.

A “cycloalkyl” group refers to an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereinone of more of the rings does not have a completely conjugatedpi-electron system. Examples, without limitation, of cycloalkyl groupsare cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane. Acycloalkyl group may be substituted or unsubstituted. When substituted,the substituent group can be, for example, alkyl, alkenyl, alkynyl,aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro,azide, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, oxo,carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,sulfonamido, and amino, as these terms are defined herein.

An “alkenyl” group refers to an alkyl group which consists of at leasttwo carbon atoms and at least one carbon-carbon double bond.

An “alkynyl” group refers to an alkyl group which consists of at leasttwo carbon atoms and at least one carbon-carbon triple bond.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. Examples,without limitation, of aryl groups are phenyl, naphthalenyl andanthracenyl. The aryl group may be substituted or unsubstituted. Whensubstituted, the substituent group can be, for example, alkyl, alkenyl,alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy,alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl,sulfonyl, cyano, nitro, azide, sulfonyl, sulfinyl, sulfonamide,phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as these termsare defined herein.

A “heteroaryl” group refers to a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl groups include pyrrole, furane,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline and purine. The heteroaryl group may besubstituted or unsubstituted. When substituted, the substituent groupcan be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro,azide, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, oxo,carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,sulfonamido, and amino, as these terms are defined herein.

A “heteroalicyclic” group refers to a monocyclic or fused ring grouphaving in the ring(s) one or more atoms such as nitrogen, oxygen andsulfur. The rings may also have one or more double bonds. However, therings do not have a completely conjugated pi-electron system. Theheteroalicyclic may be substituted or unsubstituted. When substituted,the substituted group can be, for example, lone pair electrons, alkyl,alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo,hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy,sulfinyl, sulfonyl, cyano, nitro, azide, sulfonyl, sulfinyl,sulfonamide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea,thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as theseterms are defined herein. Representative examples are piperidine,piperazine, tetrahydro furane, tetrahydropyrane, morpholino and thelike.

A “hydroxy” group refers to an —OH group.

An “azide” group refers to a —N═N group.

An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group,as defined herein.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group,as defined herein.

A “thiohydroxy” group refers to a —SH group.

A “thioalkoxy” group refers to both an —S-alkyl group, and an—S-cycloalkyl group, as defined herein.

A “thioaryloxy” group refers to both an —S-aryl and an —S-heteroarylgroup, as defined herein.

A “carbonyl” group refers to a —C(═O)—R group, where R is hydrogen,alkyl, alkenyl, cycloalkyl, aryl, heteroaryl (bonded through a ringcarbon) or heteroalicyclic (bonded through a ring carbon) as definedherein.

An “aldehyde” group refers to a carbonyl group, where R is hydrogen.

A “thiocarbonyl” group refers to a —C(═S)—R group, where R is as definedherein.

A “C-carboxy” group refers to a —C(═O)—O—R groups, where R is as definedherein.

An “O-carboxy” group refers to an RC(═O)—O— group, where R is as definedherein.

An “oxo” group refers to a ═O group.

A “carboxylic acid” group refers to a C-carboxyl group in which R ishydrogen.

A “halo” group refers to fluorine, chlorine, bromine or iodine.

A “trihalomethyl” group refers to a —CX₃ group wherein X is a halo groupas defined herein.

A “sulfinyl” group refers to an —S(═O)—R group, where R is as definedherein.

A “sulfonyl” group refers to an —S(═O)₂—R group, where R is as definedherein.

An “S-sulfonamido” group refers to a —S(═O)₂—NR₂ group, with each of Ras is defined herein.

An “N-sulfonamido” group refers to an RS(═O)₂—NR group, where each of Ris as defined herein.

An “O-carbamyl” group refers to an —OC(═O)—NR₂ group, where each of R isas defined herein.

An “N-carbamyl” group refers to an ROC(═O)—NR— group, where each of R isas defined herein.

An “O-thiocarbamyl” group refers to an —OC(═S)—NR₂ group, where each ofR is as defined herein.

An “N-thiocarbamyl” group refers to an ROC(═S)NR— group, where each of Ris as defined herein.

An “Amino” group refers to an —NR₂ group where each of R is as definedherein.

A “C-amido” group refers to a —C(═O)—NR₂ group, where each of R is asdefined herein.

An “N-amido” group refers to an RC(═O)—NR— group, where each of R is asdefined herein.

An “urea” group refers to an —NRC(═O)—NR₂ group, where each of R is asdefined herein.

A “guanidino” group refers to an —RNC(═N)—NR₂ group, where each of R isas defined herein.

A “guanyl” group refers to an R₂NC(═N)— group, where each of R is asdefined herein.

A “nitro” group refers to an —NO₂ group.

A “cyano” group refers to a —C—N group.

The term “phosphonyl” or “phosphonate” describes a —P(═O)(OR)₂ group,with R as defined hereinabove. The term “phosphate” describes an—O—P(═O)(OR)₂ group, with each of R as defined hereinabove.

A “phosphoric acid” is a phosphate group is which each of R is hydrogen.

The term “phosphinyl” describes a —PR₂ group, with each of R as definedhereinabove.

The term “thiourea” describes a —NR—C(═S)—NR— group, with each of R asdefined hereinabove.

The term “saccharide” refers to one or more sugar unit, either anopen-chain sugar unit or a cyclic sugar unit (e.g., pyranose- orfuranose-based units), and encompasses any monosaccharide, disaccharideand oligosaccharide, unless otherwise indicated.

As is shown in the general formula I above, the compounds according tothe present invention include a backbone of 1-6 carbon atoms, whereby atleast one of these backbone carbon atoms is covalently attached tohydrogen, a hydrocarbon group (alkyl, aryl, etc.), a carboxy group(e.g., acyl, carboxylic acid, etc.) or to a phosphoryl group (which isalso referred to herein as a phosphate group or simply as a phosphate),and the other 1-5 backbone carbon atoms are covalently attached tohydrocarbon chains (X₁-Xn−1) via a heteroatom (B₁-Bn in the generalformula I above). These hydrocarbon chains can include saturated orunsaturated, substituted or unsubstituted chains, optionally interruptedby aromatic, alicyclic, heteroalicyclic and/or heteroaromatic moieties,all as described hereinabove and depicted in general formula II, wherebyat least one of these chains is terminating with an oxidized group,defined hereinabove as Z that is different than hydrogen.

As used herein, the term “hydrocarbon” refers to a compound thatincludes hydrogen atoms and carbon atoms, covalently attachedtherebetween. When the hydrocarbon is saturated, each of Ca-Cm iscovalently attached to its neighboring atoms via a single sigma bond.When the hydrocarbon is unsaturated, at least two neighboring atoms ofCa-Cm are attached therebetween via a double bond or a triple bond.

Each of the hydrocarbon chains according to the present invention caninclude between 1 and 26 carbon atoms, more preferably between 3 and 26carbon atoms. Hydrocarbon chains that terminate with the oxidized groupZ are typically lower-sized chains, preferably having between 3 and 10carbon atoms, more preferably between 3 and 6 carbon atoms, notincluding the carbon atom in the oxidized group.

LDL is a lipoprotein composed of functionally different moieties(components). Among these moieties are phospholipids, which areconsidered to play an important role in the effect of oxidized LDL onplaque related diseases.

As used herein throughout, the term “moiety” or “component” refers to amajor portion of a functional molecule which is linked to anothermolecule, while retaining its activity. Phospholipids are naturalsubstances that include a non-polar lipid group and a highly polarphosphatidyl group at the end. The most prevalent phospholipids innature are phosphoglycerides, which include a glycerol backbone andfatty acyl moieties attached thereto. Phosphoglycerides such as1,2-O-fatty acyl phosphoglycerides, as well as oxidative modificationsthereof such as POVPC and PGPC, have been involved in atherogenesisrelated studies, as is described in detail hereinabove.

In addition to LDL, phospholipids and phosphoglycerides, other lipidsare involved in various biological processes such as inflammation. Theseinclude, for example, sphingolipids, glycolipids and other membranelipids.

The compounds of the present invention described above have beenprimarily designed according to the basic structure ofphosphoglycerides, such that in a preferred embodiment of the presentinvention n in the general formula I above equals 3. Such compounds arereferred to herein as oxidized phosphoglyceride analogs, while thecompounds of the present invention are collectively referred to hereinas oxidized lipids and include analogs and derivatives thereof.

As used herein throughout, the term “analogs” refers to compounds thatare structurally related to the subject molecule (e.g., oxidizedphospholipids, oxidized LDL, etc.) and can therefore exert the samebiological activity.

The term “derivatives” refers to subject molecules which has beenchemically modified but retain a major portion thereof unchanged, e.g.,subject molecules which are substituted by additional or differentsubstituents, subject molecules in which a portion thereof has beenoxidized or hydrolysed, and the like.

In view of the inherent instability of the O-fatty acyl moiety innaturally occurring phosphoglycerides, as well as in other structurallyrelated compounds, which results from its high susceptibility to fasthydrolysis in biological systems by phospholipase A₂ (see, for example,“A Textbook of Drug Design and Development”, Povl Krogsgaard-Larsen andHans Bundgaard, eds., Harwood Academic Publishers, chapter 13, pages478-480), the compounds of the present invention have been designed toinclude at least one O-fatty ether moiety, such that in the generalformula I above, when n equals 3, at least one of A₁ and A₂ ispreferably a CR″R′″ group. Compounds in which one of A₁ and A₂ is aCR″R′″ group are referred to herein as mono-etherified phosphoglycerideanalogs, while compounds in which both A₁ and A₂ are CR″R′″ are referredto herein as di-etherified phosphoglyceride analogs, and arecharacterized by improved in vivo stability, particularly as comparedwith the presently known synthetic oxidized phosphoglycerides (e.g.,POVPC and PGPC).

As is defined under the general formula I above, when n equals 3, atleast one of X₁ and X₂ is a hydrocarbon chain that terminates with anoxidized group, such that Z is not hydrogen. However, since in naturallyoccurring oxidized LDL derivatives the oxidized alkyl chain is typicallylocated at the second position, and since it has been demonstrated thatthe biological activity of several phospholipids directly depends on thestructure thereof (see the Background section for a detaileddiscussion), in another preferred embodiment of the present invention,X₂ is a hydrocarbon chain that terminates with an oxidized group.

As is further described in the general formula II hereinabove, theoxidized group can be, for example,

as well as derivatives thereof such as, for example, any carboxy orthiocarboxy derivative (e.g., a carboxylic ester in which W is oxygenand R″ is an alkyl, aryl, cycloalkyl and the like), as definedhereinabove, imino derivatives (in which W in a nitrogen atom), amidoderivatives (in which W is oxygen and R″ is an amine), phosphine orphosphonate derivatives and many more, as defined hereinabove.

One example of a novel etherified oxidized phosphoglyceride according tothe present invention is 2,5′-Aldehyde Lecithin Ether (ALLE):1-hexadecyl-2-(5′-oxo-pentanyl)-sn-glycero-3-phosphocholine(D-ALLE),3-hexadecyl-2-(5′-oxo-pentanyl)-sn-glycero-1-phosphocholine(L-ALLE)], and the racemic mixture thereof, the synthesis and use ofwhich are further detailed in the Examples section which follows.

However, as aldehydes are known as unstable compounds, which tend to beeasily oxidized, preferred examples of novel etherified oxidizedphosphoglycerides according to the present invention include the acidderivative 1-Hexadecyl-2-(5′-Carboxy-butyl)-sn-glycero-3-phosphocholine(also referred to hereinafter as IC-201), and its corresponding acetals1-Hexadecyl-2-(5′,5′-Dimethoxy-pentyloxy)-sn-glycero-3-phosphocholineand 1-Hexadecyl-2-(5′,5′-Diethoxy-pentyloxy)-sn-glycero-3-phosphocholine(see FIG. 10 for 2-D structural formulas), the synthesis and use ofwhich are also further detailed in the Examples section which follows.

While the oxidized lipids described above are derived fromphosphoglycerides, oxidized lipids derived from, for example,sphingolipids, are also within the scope of the present invention. Suchoxidized sphingolipids analogs according to the present invention havethe general formula I above, wherein n equals 3, Y is hydrogen, B₂ isNH, and A₂ is C═O, whereby the hydrocarbon chain terminating with anoxidized group is attached either to the amide, as X₂ or to C₁.

While oxidized phosphoglycerides are derived from glycerol, which is amonosaccharide molecule, and oxidized sphingolipids are derived fromsphingosine, an amino alcohol, it is envisioned that oxidizedphospholipids derived from other biologically prevalent alcohol baseunits would exert the same effect. Furthermore, since no correlationbetween the distance of the oxidized moiety and the phosphatidyl moietyin oxidized phospholipids has been established, it is envisioned thatoxidized lipids that are derived from a 4-6 carbon atoms backbone wouldretain structure characteristics similar to those of oxidizedphosphoglycerides and as such in all probability would possess the sameantigenicity and immune modulation activity, and employed and appliedsimilarly to the oxidized phosphoglyceride derivatives described herein.

A preferred example of such an alcohol base unit is a monosaccharidebase unit, such as, for example, glucose, erythritol and threitol.

Thus, in another preferred embodiment, the compounds according to thepresent invention include up to 6 carbon atoms in the backbone chain.The carbon atoms in the backbone chain can be linearly attached one toanother, so as to form an open-chain monosaccharide backbone, oralternatively, can form a heteroalicyclic monosaccharide backbone,namely a pyranose or furanose backbone, such that in the general formulaabove, one of R₁ and R′₁, is covalently attached to one of Rn or R′n,via an etheric bond (an R—O—R bond)

Still alternatively, the compounds of the present invention can include4-6 carbon atoms in the backbone chain, which form a non-saccharidicring, namely a four-, five- or six-membered carbocyclic orheteroalicyclic ring, such that in the general formula I above one of R₁and R′₁, is covalently attached to one of Rn or R′n, via different bonds(e.g., a sigma bond, a π bond, a carboxylic bond, an ether bond, athioether bond and any other bond).

As is further described in the general formula I hereinabove, Y iseither a phosphoryl moiety (e.g., phosphoryl choline, phosphorylethanolamine, etc.) or a non-phosphoryl moiety (e.g., hydrogen, acyl oralkyl). When Y is a non-phosphoryl moiety, the resultant compound is nota phospholipid, rather a diglyceride compound, for n=3, or any otheralcohol-derived, e.g., monosaccharide-derived compound.

Since no particular activity of the phosphoryl group has been taught sofar with respect to the immunomodulation activity of oxidized LDL, it isfurther envisioned that such non-phosphoryl compounds would retainsimilar structure characteristics as the above oxidized phospholipidsand as such in all probability would posses antigenicity and immunemodulation activity, and can be employed and applied similarly to theoxidized phospholipid derivatives described herein.

In an embodiment of the present invention, Y is a saccharide, as isdefined hereinabove, and thus the compound according to the presentinvention is an oxidized analog of glycolipids.

In another embodiment, the compound is an oxidized analog of anymembrane lipid.

The preferred structural features described above with respect tooxidized phosphoglycerides apply for all the compounds describedhereinabove. Hence, in a preferred embodiment of the present invention,at least one of A₁, A₂, . . . and An−1 is a CR″R′″ group, such that thecompound include at least one etherified side chain. Due to theinstability of an O-acyl side chain, it is further preferred that atleast one of the oxidized groups in X₁-Xn−1 would be linked to such anetherified side chain.

Although naturally occurring phospholipids and oxidized phospholipidstypically include O-acyl chains, there is evidence that thiolderivatives of oxidized phospholipids, which include, for example,S-acyl chains, may exert the same biological activity (see, for example,Reddy et al. Antitumor ether lipids: an improved synthesis of ilmofosineand an entioselective synthesis of an ilmofosine analog. TetrahedronLetters. 1994; 17:2679-2682; Batia and Hajdu. Stereospecific synthesisof ether and thioether phospholipids. The use of L-glyceric acid as achiral phospholipids precursor. J. Org. Chem. 1988; 53:5034-5039; Bosieset al. Lipids. 1987; 22:947; Bosies et al. Ger. Offen. DE 3,906,952[C.A. 1991, 114, 102394w]; and Herrmann et al. NCI-EORTC Symposium onNew Drugs in Cancer Therapy, Amsterdam, March 1992). As thiols arecharacterized by enhanced biostability, such compounds can further behighly beneficial.

Hence, in one embodiment of the present invention, at least one of B₁-Bnis sulfur, such that at least one of the side chains is a thiolatedS-acyl or an s-alkyl chain. In another embodiment, at least one ofX₁-Xn−1 which comprises an oxidized group is linked to such a thiolatedside chain.

Alternatively, each of B₁-Bn can be a biocompatible heteroatom otherthan oxygen and sulfur, such as, for example, nitrogen, phosphor orsilicon, as is described within the general formula I hereinabove.

Apart from the structural features delineated herein, the compounds ofthe present invention can be further substituted at any positionthereof, e.g., at any of the side chain carbon atoms and at any of thebackbone carbon atoms. While a myriad of possible substituentsdelineated hereinabove and encompassed by the present invention,preferred substituents include, for example, halo and aryl.

Although the compounds of the present invention have been basicallydesigned from oxidized phospholipids such as phosphoglycerides, thepresent inventors also envisage that a single oxidized hydrocarbonchain, which is optionally attached to a polar group, would exert thatsame antigenicity and immunomodulation activity as the oxidizedphospholipid analogs described above.

Such an oxidized hydrocarbon chain is a common feature of arachidonicacid metabolites. Arachidonic acid is a polyunsaturated fatty acidhaving 20 carbon atoms, which is produced in vivo by the enzymatichydrolysis of phospholipids containing same. Upon its release,arachidonic acid is oxidized into a number of important autacoids bycertain lipoxygenases and following a cascade of additional enzymaticreactions, the autacoids are metabolized into a family of classicalprostaglandins (PG), prostacyclin (PGI₂) and thromboxane (TX) A₂, whichare active in many biological pathways. All these metabolites include acommon feature of a six-carbon chain terminating with an oxidizabledouble bond.

As is described hereinabove and is further demonstrated in the Examplessection that follows, the presence of an oxidized group in oxidized LDLanalogs that are designed for mimicking the immunomodulation induced byox LDL, is essential. Thus, in comparative studies it was shown, forexample, that Compound V, the non-oxidized compound corresponding toCI-201 (Compound VII), is non-active while CI-201 is (see, for example,Examples XIV and XV in the Examples section that follows). Furthermore,based on the metabolism pathway of arachidonic acid, it is assumed thatother oxidized phospholipids undergo the same pathway, which results inthe release of the oxidized side chain. As is further describedhereinabove, the oxidized side chain preferably includes between 3 and 7carbon atoms, and is therefore similar to the six-carbon chain featureon the arachidonic acid metabolites. Moreover, the CD1 mechanismdescribed above, which suggest a role for lipids in the immune system,indicate that the hydrophilic head, i.e. the carbon-C2 and/or thecarbon-C3 head group in CD1-d are most probably the antigenic epitopepresented to the immune system as it is the part presenting by the CD1groove that hide the hydrophobic part of the molecule, indicating a roleof an hydrophilic epitope at carbon-C2.

In oxidized phospholipids such as phosphoglycerides, the oxidized sidechain is attached to a phosphoglycerol backbone. However, as ismentioned hereinabove, no particular role for the phosphoglycerolbackbone has been suggested.

Hence, in a preferred embodiment of the present invention, n equals 1,such that the compound of the present invention is a single hydrocarbonchain terminating with an oxidized group. While such an oxidized singlehydrocarbon chain is non-polar, it can be attached to a polar group suchas a phosphoryl group, such that in the general formula I hereinabove,when n equals 1, at least one of R₁ and R′₁ is a phosphate orphosphonate group. Alternatively, at least one of R₁ and R′₁ can beselected from other biocompatible polar groups such as, for example,peptides, saccharides and the like.

Depending on the substituents, each of the carbon atoms in each of thecompounds described above, namely C₁-Cn and Ca-Cm, can be chiral ornon-chiral. Any chiral carbon atom that is present in the compounds ofthe present invention can be either in an R-configuration, anS-configuration or racemic. Thus the present invention encompasses anycombination of chiral and racemic carbon atoms, including all thepossible stereoisomers, optical isomers, enantiomers, and anomers. As isdemonstrated in the Examples section that follows, the compounds of thepresent invention can be synthesized while retaining a configuration ofthe starting material. The compounds of the present invention can befurther selectively synthesized in terms of the stereochemistry of theoxidized group. Hence, by selecting the appropriate starting materialsand the appropriate syntheses conditions, the optical purity (e.g., theinclusion of chiral and/or racemic carbons) and the obtainedstereoisomers of the resulting compounds can be determined. In caseswhere racemic mixtures are obtained, known techniques can be used toseparate the optical or stereo-isomers. Such techniques are described,for example, in “Organic chemistry, fourth Edition by Paula YurkanisBruice, page 180-185 and page 214, Prentice Hall, Upper Sadde River,N.J. 07458”.

The present invention further encompasses any pharmaceuticallyacceptable salts, prodrugs, hydrates and solvates of the compoundsdescribed hereinabove.

The term “prodrug” refers to an agent, which is converted into theactive compound (the active parent drug) in vivo. Prodrugs are typicallyuseful for facilitating the administration of the parent drug. They may,for instance, be bioavailable by oral administration whereas the parentdrug is not. The prodrug may also have improved solubility as comparedwith the parent drug in pharmaceutical compositions. Prodrugs are alsooften used to achieve a sustained release of the active compound invivo. An example, without limitation, of a prodrug would be a compoundof the present invention, having one or more carboxylic acid moieties,which is administered as an ester (the “prodrug”). Such a prodrug ishydrolysed in vivo, to thereby provide the free compound (the parentdrug). The selected ester may affect both the solubility characteristicsand the hydrolysis rate of the prodrug.

The phrase “pharmaceutically acceptable salt” refers to a chargedspecies of the parent compound and its counter ion, which is typicallyused to modify the solubility characteristics of the parent compoundand/or to reduce any significant irritation to an organism by the parentcompound, while not abrogating the biological activity and properties ofthe administered compound. An example, without limitation, of apharmaceutically acceptable salt would be a carboxylate anion and acation such as, but not limited to, ammonium, sodium, potassium and thelike.

The term “solvate” refers to a complex of variable stoichiometry (e.g.,di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by asolute (the compound of present invention) and a solvent, whereby thesolvent does not interfere with the biological activity of the solute.Suitable solvents include, for example, ethanol, acetic acid and thelike.

The term “hydrate” refers to a solvate, as defined hereinabove, wherethe solvent is water.

As is detailed hereinbelow, the newly designed compounds of the presentinvention exert a highly beneficial immunomodulation activity andtherefore can be utilized in various therapeutic applications. Utilizingthese compounds in therapeutic application involves administrationthereof either per se, or as a part of a pharmaceutical compositionwhere it is mixed with suitable carriers or excipients.

Thus, according to another aspect of the present invention, there isprovided a pharmaceutical composition, which comprises, as an activeingredient, any of the compounds described hereinabove in generalformula I and the accompanying description, and a pharmaceuticallyacceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the compounds (e.g., ALLEand CI-201 and other compounds depicted in the general formula Ihereinabove) accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

In a preferred embodiment of the present invention, the pharmaceuticalcompositions are designed for modulating an immune and/or inflammatoryresponse via mucosal administration.

In another preferred embodiment of the present invention, thepharmaceutical compositions are designed modulating an immune and/orinflammatory response via oral administration.

Further preferably, the pharmaceutical compositions of the presentinvention are designed for nasal, or intraperitoneal administration, asis detailed hereinafter.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of a disorder (e.g., atherosclerosis) or prolong the survivalof the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, e.g., Fingl, et al., 1975, in “The PharmacologicalBasis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma or brain levels of the active ingredient are sufficient to induceor suppress angiogenesis (minimal effective concentration, MEC). The MECwill vary for each preparation, but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. Detection assays can beused to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed hereinbelow.

Thus, in a preferred embodiment of the present invention, thepharmaceutical composition is packaged in a packaging material andidentified in print, on or in the packaging material, for use in thetreatment or prevention of an inflammation associated with an endogenousoxidized lipid. A list of representative examples of diseases anddisorders associated with such an inflammation is provided hereinbelow.

As is further described in detail hereinbelow, the pharmaceuticalcomposition of the present invention can further include an additionalcompound, which is useful in the treatment or prevention of the aboveinflammation.

As is described in detail in the Examples section that follows,representative examples of the newly designed compounds of the presentinvention have been found effective in modulating an immune responseand/or an inflammatory response associated with endogenous oxidized LDL,thus leading to attenuation of diseases associated with endogenousoxidized LDL. These results clearly suggest that (i) modulation of animmune and/or inflammatory response to endogenous oxidized LDL inparticular and endogenous oxidized lipids in general can be induced byany compound that is structurally related to an oxidized lipid; and (ii)compounds capable of modulating an immune and/or inflammatory responseto oxidized lipids can be utilized to treat or prevent inflammationassociated with endogenous oxidized lipids.

Hence, according to another aspect of the present invention there isprovided a method of treating or preventing an inflammation associatedwith an endogenous oxidized lipid. The method according to this aspectof the present invention is effected by administering to a subject inneed thereof a therapeutically effective amount of one or more oxidizedlipids.

As used herein, the phrase “an endogenous oxidized lipid” refers to oneor more oxidized lipids that are present or formed in vivo, as a resultof inflammatory and other cell- or humoral-mediated processes.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures bypractitioners of the chemical, pharmacological, biological, biochemicaland medical arts.

As used herein, the phrase “treating or preventing” includes abrogating,substantially inhibiting, slowing or reversing the progression of adisease, substantially ameliorating clinical symptoms of a disease orsubstantially preventing the appearance of clinical symptoms of adisease.

Examples of subjects suitable for such treatment include subjectssuffering from a disease or disorder associated with an inflammation, asis detailed hereinbelow. Preferred individual subjects according to thepresent invention are mammals such as canines, felines, ovines,porcines, equines, and bovines. Preferably the individual subjectsaccording to the present invention are humans.

The phrase “oxidized lipid” refers to a natural or, preferably,synthetically prepared, compound that has common structural featureswith a natural lipid, an oxidized lipid, and any components, moieties,analogs and derivatives thereof. For example, oxidized LDL is composedof several functionally and structurally different moieties, and thisphrase encompasses any synthetically prepared compound that has commonstructural features with any one of these moieties. This phrase furtherencompasses any derivative of such analogs.

Representative examples of oxidized lipids include, without limitation,oxidized phospholipids, platelet activating factor analogs, plasmalogenanalogs, substituted or unsubstituted 3-30 carbon atoms hydrocarbonsterminating with an oxidized group, sphingolipids oxidized analogs,glycolipids oxidized analogs, oxidized analogs of membrane lipids andany analogs or derivatives thereof.

Phospholipids in general and phosphoglycerides in particular are wellknown lipids, which are also components of oxidized LDL.Phosphoglycerides are derivatives of phsophoglycerol, which include oneor more fatty acyl or acyl groups attached to the phsophoglycerolbackbone.

Hence, synthetically prepared oxidized phospholipids may be efficientlyused in the method according to this aspect of the present invention.Representative examples of known synthetic oxidized phospholipidsinclude, without limitation,1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC),1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), and1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphocholine.

More preferred examples of synthetic oxidized phospholipids include thecompounds of the present invention, as described hereinabove, includingthose compounds in which n=1. The latter are delineated herein assubstituted or unsubstituted 3-30 carbon atoms hydrocarbons terminatingwith an oxidized group.

Other compounds which are structurally related to oxidizedphosphoglycerides, and can therefore be efficiently used in this andother aspects of the present invention, are platelet-activating factor(PAF) analogs.

PAF are 1-alkyl-2-acetyl-sn-glycero-3-phosphocholines, naturallyoccurring ether-linked glycerolipids. The alkyl chain at the sn−1position is typically an unsaturated alkyl having 16-18 carbon atoms.Some well-known PAF analogs typically include substitution of the acylmoiety at the sn−2 position by a long-chain acyl moiety (e.g., a fattyacid acyl). Additional PAF analogs include an oxidative modification,either at the unsaturated O-alkyl chain present in the sn−1 position orat the fatty acyl chain present at the sn−2 position.

Representative examples of known PAF analogs that can be used in thiscontext of the present invention include, without limitation,1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphocholine,1-hexadecyl-2-acetoyl-sn-glycero-3-phosphocholine,1-octadecyl-2-acetoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-butyroyl-sn-glycero-3-phosphocholine,1-octadecyl-2-butyroyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine,1-octadecenyl-2-acetoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-(homogammalinolenoyl)-sn-glycero-3-phosphocholine,1-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-eicosapentaenoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine,1-octadecyl-2-methyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-butenoyl-sn-glycero-3-phosphocholine, Lyso PAF C16 andLyso PAF C18. However, any other PAF analogs or derivatives thereof canfurther be used in this context of the present invention.

Additional compounds, which are structurally related to oxidizedphosphoglycerides, and can therefore be efficiently used in this andother aspects of the present invention, are plasmalogen analogs.

Plasmalogens are 1-alkyl-2-acetyl-sn-glycero-3-phosphatidyl, naturallyoccurring ether-linked glycerolipids, in which the alkyl chain at thesn−1 position is typically saturated. Some well-known plasmalogenanalogs typically include substitution of the acyl moiety at the sn−2position by a long-chain acyl moiety (e.g., a fatty acid acyl) andfurther include an oxidative modification, either at the sn−1 positionor at the sn−2 position.

Representative examples of known plasmalogen analogs that can be used inthis context of the present invention include, without limitation,1-O-1′-(Z)-hexadecenyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine,1-O-1′-(Z)-hexadecenyl-2-oleoyl-sn-glycero-3-phosphocholine,1-O-1′-(Z)-hexadecenyl-2-arachidonoyl-sn-glycero-3-phosphocholine,1-O-1′-(Z)-hexadecenyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine,1-O-1′-(Z)-hexadecenyl-2-oleoyl-sn-glycero-3-phosphoethanolamine,1-O-1′-(Z)-hexadecenyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine,and1-O-1′-(Z)-hexadecenyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine.However, any other plasmalogen analogs or derivatives thereof canfurther be used in this context of the present invention.

As used herein, the phrase “an inflammation associated with anendogenous oxidized lipid” describes an inflammation that is associatedwith the in vivo formation or presence of one or more oxidized lipids(e.g., oxidized LDL, oxidized membrane lipids, etc.).

Inflammation is a protective response of the body to an injury. Severalcytokines play key roles in mediating inflammatory reactions amongst areIFN-γ and IL-10. IFN-γ has been implicated in the pathogenesis of avariety of autoimmune and chronic inflammatory conditions. On the otherhand, IL-10 inhibits IFN-γ production by activated immune cells such asTH2 and M2 cells this cytokine (IL-10) serve as the majoranti-inflammatory “gate”.

Excessive inflammation is oftentimes deleterious, involving or leadingto a myriad of diseases and disorders. As is explained in detailhereinabove, excessive inflammatory response is typically associatedwith oxidized lipid epitopes.

As is shown in the Examples section that follows, modulating the immuneresponse to oxidized LDL by synthetic oxidized LDL analogs is associatedwith an anti-inflammatory effect. This anti-inflammatory effect may beutilized in treating or preventing inflammation-associated disease ordisorders in which endogenous oxidized LDL or any other endogenousoxidized lipid is implicated. Such diseases and disorders include, forexample, diseases or disorders associated with plaque formation,including but not limited to atherosclerosis, atheroscleroticcardiovascular disease, cerebrovascular disease, peripheral vasculardisease, stenosis, restenosis and in-stent-stenosis, as well asautoimmune diseases or disorders, neurodegenerative diseases ordisorders, proliferative disease or disorders and aging processes.

Thus, representative examples of diseases or disorders associated withan inflammation, which in turn is associated with an endogenous oxidizedlipids, and are therefore treatable by the method of the presentinvention include, for example, idiopathic inflammatory diseases ordisorders, chronic inflammatory diseases or disorders, acuteinflammatory diseases or disorders, autoimmune diseases or disorders,infectious diseases or disorders, inflammatory malignant diseases ordisorders, inflammatory transplantation-related diseases or disorders,inflammatory degenerative diseases or disorders, diseases or disordersassociated with a hypersensitivity, inflammatory cardiovascular diseasesor disorders, inflammatory cerebrovascular diseases or disorders,peripheral vascular diseases or disorders, inflammatory glandulardiseases or disorders, inflammatory gastrointestinal diseases ordisorders, inflammatory cutaneous diseases or disorders, inflammatoryhepatic diseases or disorders, inflammatory neurological diseases ordisorders, inflammatory musculo-skeletal diseases or disorders,inflammatory renal diseases or disorders, inflammatory reproductivediseases or disorders, inflammatory systemic diseases or disorders,inflammatory connective tissue diseases or disorders, inflammatorytumors, necrosis, inflammatory implant-related diseases or disorders,inflammatory aging processes, immunodeficiency diseases or disorders,proliferative diseases and disorders and inflammatory pulmonary diseasesor disorders, as is detailed hereinbelow.

Non-limiting examples of hypersensitivities include Type Ihypersensitivity, Type II hypersensitivity, Type III hypersensitivity,Type IV hypersensitivity, immediate hypersensitivity, antibody mediatedhypersensitivity, immune complex mediated hypersensitivity, T lymphocytemediated hypersensitivity, delayed type hypersensitivity, helper Tlymphocyte mediated hypersensitivity, cytotoxic T lymphocyte mediatedhypersensitivity, TH1 lymphocyte mediated hypersensitivity, and TH2lymphocyte mediated hypersensitivity.

Non-limiting examples of inflammatory cardiovascular disease or disorderinclude occlusive diseases or disorders, atherosclerosis, a cardiacvalvular disease, stenosis, restenosis, in-stent-stenosis, myocardialinfarction, coronary arterial disease, acute coronary syndromes,congestive heart failure, angina pectoris, myocardial ischemia,thrombosis, Wegener's granulomatosis, Takayasu's arteritis, Kawasakisyndrome, anti-factor VIII autoimmune disease or disorder, necrotizingsmall vessel vasculitis, microscopic polyangiitis, Churg and Strausssyndrome, pauci-immune focal necrotizing glomerulonephritis, crescenticglomerulonephritis, antiphospholipid syndrome, antibody induced heartfailure, thrombocytopenic purpura, autoimmune hemolytic anemia, cardiacautoimmunity, Chagas' disease or disorder, and anti-helper T lymphocyteautoimmunity.

Stenosis is an occlusive disease of the vasculature, commonly caused byatheromatous plaque and enhanced platelet activity, most criticallyaffecting the coronary vasculature.

Restenosis is the progressive re-occlusion often following reduction ofocclusions in stenotic vasculature. In cases where patency of thevasculature requires the mechanical support of a stent,in-stent-stenosis may occur, re-occluding the treated vessel.

Non-limiting examples of cerebrovascular diseases or disorders includestroke, cerebrovascular inflammation, cerebral hemorrhage and vertebralarterial insufficiency.

Non-limiting examples of peripheral vascular diseases or disordersinclude gangrene, diabetic vasculopathy, ischemic bowel disease,thrombosis, diabetic retinopathy and diabetic nephropathy.

Non-limiting examples of autoimmune diseases or disorders include all ofthe diseases caused by an immune response such as an autoantibody orcell-mediated immunity to an autoantigen and the like. Representativeexamples are chronic rheumatoid arthritis, juvenile rheumatoidarthritis, systemic lupus erythematosus, scleroderma, mixed connectivetissue disease, polyarteritis nodosa, polymyositis/dermatomyositis,Sjogren's syndrome, Bechet's disease, multiple sclerosis, autoimmunediabetes, Hashimoto's disease, psoriasis, primary myxedema, perniciousanemia, myasthenia gravis, chronic active hepatitis, autoimmunehemolytic anemia, idiopathic thrombocytopenic purpura, uveitis,vasculitides and heparin induced thrombocytopenia.

Non-limiting examples of inflammatory glandular diseases or disordersinclude pancreatic diseases or disorders, Type I diabetes, thyroiddiseases or disorders, Graves' disease, thyroiditis, spontaneousautoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema,ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmuneprostatitis and Type I autoimmune polyglandular syndrome.

Non-limiting examples of inflammatory gastrointestinal diseases ordisorders disorders include colitis, ileitis, Crohn's disease, chronicinflammatory intestinal disease, inflammatory bowel syndrome, chronicinflammatory bowel disease, celiac disease, ulcerative colitis, anulcer, a skin ulcer, a bed sore, a gastric ulcer, a peptic ulcer, abuccal ulcer, a nasopharyngeal ulcer, an esophageal ulcer, a duodenalulcer and a gastrointestinal ulcer.

Non-limiting examples of inflammatory cutaneous diseases or disordersdisorders include acne, and an autoimmune bullous skin disease.

Non-limiting examples of inflammatory hepatic diseases or disordersinclude autoimmune hepatitis, hepatic cirrhosis, and biliary cirrhosis.

Non-limiting examples of inflammatory neurological diseases or disordersinclude multiple sclerosis, Alzheimer's disease, Parkinson's disease,myasthenia gravis, motor neuropathy, Guillain-Barre syndrome, autoimmuneneuropathy, Lambert-Eaton myasthenic syndrome, paraneoplasticneurological disease or disorder, paraneoplastic cerebellar atrophy,non-paraneoplastic stiff man syndrome, progressive cerebellar atrophy,Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea,Gilles de la Tourette syndrome, autoimmune polyendocrinopathy, dysimmuneneuropathy, acquired neuromyotonia, arthrogryposis multiplex,Huntington's disease, AIDS associated dementia, amyotrophic lateralsclerosis (AML), multiple sclerosis, stroke, an inflammatory retinaldisease or disorder, an inflammatory ocular disease or disorder, opticneuritis, spongiform encephalopathy, migraine, headache, clusterheadache, and stiff-man syndrome.

Non-limiting examples of inflammatory connective tissue diseases ordisorders include autoimmune myositis, primary Sjogren's syndrome,smooth muscle autoimmune disease or disorder, myositis, tendinitis, aligament inflammation, chondritis, a joint inflammation, a synovialinflammation, carpal tunnel syndrome, arthritis, rheumatoid arthritis,osteoarthritis, ankylosing spondylitis, a skeletal inflammation, anautoimmune ear disease or disorder, and an autoimmune disease ordisorder of the inner ear.

Non-limiting examples of inflammatory renal diseases or disordersinclude autoimmune interstitial nephritis and/or renal cancer.

Non-limiting examples of inflammatory reproductive diseases or disordersinclude repeated fetal loss, ovarian cyst, or a menstruation associateddisease or disorder.

Non-limiting examples of inflammatory systemic diseases or disordersinclude systemic lupus erythematosus, systemic sclerosis, septic shock,toxic shock syndrome, and cachexia.

Non-limiting examples of infectious disease or disorder include chronicinfectious diseases or disorders, a subacute infectious disease ordisorder, an acute infectious disease or disorder, a viral disease ordisorder, a bacterial disease or disorder, a protozoan disease ordisorder, a parasitic disease or disorder, a fungal disease or disorder,a mycoplasma disease or disorder, gangrene, sepsis, a prion disease ordisorder, influenza, tuberculosis, malaria, acquired immunodeficiencysyndrome, and severe acute respiratory syndrome.

Non-limiting examples of inflammatory transplantation-related diseasesor disorders include graft rejection, chronic graft rejection, subacutegraft rejection, acute graft rejection hyperacute graft rejection, andgraft versus host disease or disorder. Exemplary implants include aprosthetic implant, a breast implant, a silicone implant, a dentalimplant, a penile implant, a cardiac implant, an artificial joint, abone fracture repair device, a bone replacement implant, a drug deliveryimplant, a catheter, a pacemaker, an artificial heart, an artificialheart valve, a drug release implant, an electrode, and a respiratortube.

Non-limiting examples of inflammatory tumors include a malignant tumor,a benign tumor, a solid tumor, a metastatic tumor and a non-solid tumor.

Non-limiting examples of inflammatory pulmonary diseases or disordersinclude asthma, allergic asthma, emphysema, chronic obstructivepulmonary disease or disorder, sarcoidosis and bronchitis.

An examples of a proliferative disease or disorder is cancer.

The implication of phospholipids and phospholipid metabolites intreating of preventing diseases and syndromes such as, for example,oxidative stress of aging (Onorato J M, et al, Annal N Y Acad Sci 1998Nov. 20; 854:277-90), rheumatoid arthritis (RA)(Paimela L, et al. AnnRheum Dis 1996 August; 55(8):558-9), juvenile rheumatoid arthritis(Savolainen A, et al, 1995; 24(4):209-11), inflammatory bowel disease(IBD)(Sawai T, et al, Pediatr Surg Int 2001 May; 17(4):269-74) and renalcancer (Noguchi S, et al, Biochem Biophys Res Commun 1992 Jan. 31;182(2):544-50), has been recently reported, and thus further support thebeneficial use of oxidized LDL analogs in the treatment or prevention ofthese diseases or disorders.

According to the method of the present invention, the oxidized lipidscan be administered to a subject by various routes, including, forexample, the oral, rectal, transmucosal, especially transnasal,intestinal or parenteral delivery, including intramuscular, subcutaneousand intramedullary injections as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular routes. However, as is described in detail herein throughoutand is further demonstrated in the Examples section that follows,preferred routes of administration include the oral, mucosal, nasal,intradermal (subcutaneous) and intraperitoneal routes.

Hence, in one embodiment, 0.1-100 mg/kg of an oxidized lipid isadministered intraperitoneally, in a suitable carrier such as but notlimited to PBS or glycerol, one to three times, every week, on a chronicor alternate regiment.

In another embodiment, 0.1-100 mg/kg of an oxidized lipid isadministered nasally, in a suitable carrier such as but not limited toPBS or glycerol, one to three times, every week, on a chronic oralternate regiment.

In still another embodiment, 0.1-100 mg/kg of an oxidized lipid isadministered subcutaneously, in a suitable carrier such as but notlimited to PBS or glycerol, one to three times, every week, on a chronicor alternate regiment.

In yet another embodiment, 0.1-100 mg/kg of an oxidized lipid isadministered orally, in a suitable carrier such as but not limited toPBS or glycerol, one to three times, every week, on a chronic oralternate regiment.

The pharmaceutical compositions and the method according to the presentinvention, described hereinabove, may further involve the administrationof one or more additional compounds that are capable of treating orpreventing an inflammation associated with endogenous oxidized lipid asdelineated hereinabove.

The methods according to the present invention can therefore involveco-administering, prior to, concomitant with or after the administrationof the oxidized lipids, a therapeutically effective amount of one ormore of such additional compounds, while the pharmaceutical compositionaccording to the present invention may include, in addition to thecompounds of the present invention, such additional compounds.

Representative examples of additional compounds that are capable oftreating or preventing an inflammation associated with endogenousoxidized lipid delineated hereinabove, and are therefore usable is thecontext of this embodiment of the present invention include, withoutlimitation, HMGCoA reductase inhibitors (statins), mucosal adjuvants,corticosteroids, steroidal anti-inflammatory drugs, non-steroidalanti-inflammatory drugs, analgesics, growth factors, toxins, cholesterylester transfer protein (CETP) inhibitors, perixosomes, proliferativeactivated receptor (PPAR) agonists, anti-atherosclerosis drugs,anti-proliferative agents, ezetimide, nicotinic acid, squaleninhibitors, an ApoE Milano, HSPs, Beta-2-glycoprotein-I and anyderivative and analog thereof.

HMGCoA reductase inhibitors (statins) are well known drugs thateffectively reduce LDL-cholesterol levels by inhibiting the enzyme thatregulates the rate of cholesterol production and increasing theclearance of LDL-cholesterol present in the blood by the liver.Non-limiting examples of commonly prescribed statins includeAtorvastatin, Fluvastatin, Lovastatin, Pravastatin and Simvastatin.

Ezetimibe is the first of a new class of cholesterol absorptioninhibitors that potently and selectively inhibits dietary and biliarycholesterol absorption at the brush border of the intestinal epithelium,without affecting the absorption of triglyceride or fat-solublevitamins. Ezetimibe thus reduces overall cholesterol delivery to theliver, secondarily inducing increased expression of LDL receptors,resulting in an increased removal of LDL-C from the plasma.

Peroxisome is a single-membrane organelle present in nearly alleukaryotic cells. One of the most important metabolic processes of theperoxisome is the β-oxidation of long and very long chain fatty acids.The peroxisome is also involved in bile acid synthesis, cholesterolsynthesis, plasmalogen synthesis, amino acid metabolism, and purinemetabolism.

Nicotinic acid is a known agent that lowers total cholesterol,LDL-cholesterol, and triglyceride levels, while raising HDL-cholesterollevels. There are three types of nicotinic acid drugs: immediaterelease, timed release, and extended release. Nicotinic acid or niacin,the water-soluble B vitamin, improves all lipoproteins when given indoses well above the vitamin requirement.

Squalene, an isoprenoid compound structurally similar to beta-carotene,is an intermediate metabolite in the synthesis of cholesterol. Inhumans, about 60 percent of dietary squalene is absorbed. It istransported in serum generally in association with very low densitylipoproteins and is distributed ubiquitously in human tissues, with thegreatest concentration in the skin, where it is one of the majorcomponents of skin surface lipids. Squalene inhibitors (e.g.,monooxygenase and synthase) serve as cholesterol biosynthesisinhibitors.

Proliferative Activated Receptor (PPAR) agonists, e.g., fibrates, arefatty acid-activated members of the nuclear receptor superfamily thatplay important roles in lipid and glucose metabolism, and have beenimplicated in obesity-related metabolic diseases such as hyperlipidemia,insulin resistance, and coronary artery disease. Fibrates are generallyeffective in lowering elevated plasma triglycerides and cholesterol andact as PPAR agonists. The most pronounced effect of fibrates includes adecrease in plasma triglyceride-rich lipoproteins (TRLs). Levels of LDLcholesterol (LDL-C) generally decrease in individuals with elevatedbaseline plasma concentrations, and HDL cholesterol (HDL-C) levels areusually increased when baseline plasma concentrations are low.Non-limiting examples of commonly prescribed fibrates includebezafibrate, gemfibrozil and fenofibrate.

Cholesteryl Ester Transfer Protein (CETP) inhibitors play a major rolein atherogenesis, by reducing cholesteryl ester accumulation withinmacrophages and the arterial wall, and thus reducing foam cell formationand affecting the cholesterol absorption. The most promising presentlyknown CETP inhibitor is avisimibe.

ApoA-I Milano is typically used as a recombinant complex withphospholipid (ETC-216) and produces significant regression of coronaryatherosclerosis.

Co-administration of mucosal adjuvants has been shown to be essential inpreventing the invasion of infectious agents through mucosal surfaces.In the early stages of induction of mucosal immune response, the uptakeof orally or nasally administered antigens is achieved through a uniqueset of antigen-sampling cells, M cells located in follicle-associatedepithelium (FAE) of inductive sites. After successful uptake, theantigens are immediately processed and presented by the underlyingdendritic cells (DCs).

Non-limiting examples of non-steroidal anti-inflammatory drugs includeoxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam, andCP-14,304; salicylates, such as aspirin, disalcid, benorylate,trilisate, safapryn, solprin, diflunisal, and fendosal; acetic acidderivatives, such as diclofenac, fenclofenac, indomethacin, sulindac,tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin,fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac;fenamates, such as mefenamic, meclofenamic, flufenamic, niflumic, andtolfenamic acids; propionic acid derivatives, such as ibuprofen,naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen,indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen,tioxaprofen, suprofen, alminoprofen, and tiaprofenic; pyrazoles, such asphenylbutazone, oxyphenbutazone, feprazone, azapropazone, andtrimethazone.

Non-limiting examples of steroidal anti-inflammatory drugs include,without limitation, corticosteroids such as hydrocortisone,hydroxyltriamcinolone, alpha-methyl dexamethasone,dexamethasone-phosphate, beclomethasone dipropionates, clobetasolvalerate, desonide, desoxymethasone, desoxycorticosterone acetate,dexamethasone, dichlorisone, diflorasone diacetate, diflucortolonevalerate, fluadrenolone, fluclorolone acetonide, fludrocortisone,flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortinebutylesters, fluocortolone, fluprednidene (fluprednylidene) acetate,flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisonebutyrate, methylprednisolone, triamcinolone acetonide, cortisone,cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate,fluradrenolone, fludrocortisone, difluorosone diacetate, fluradrenoloneacetonide, medrysone, amcinafel, amcinafide, betamethasone and thebalance of its esters, chloroprednisone, chlorprednisone acetate,clocortelone, clescinolone, dichlorisone, diflurprednate, flucloronide,flunisolide, fluoromethalone, fluperolone, fluprednisolone,hydrocortisone valerate, hydrocortisone cyclopentylpropionate,hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone,beclomethasone dipropionate, triamcinolone, and mixtures thereof.

Non-limiting examples of analgesics (pain relievers) include aspirin andother salicylates (such as choline or magnesium salicylate), ibuprofen,ketoprofen, naproxen sodium, and acetaminophen.

Growth factors are hormones which have numerous functions, includingregulation of adhesion molecule production, altering cellularproliferation, increasing vascularization, enhancing collagen synthesis,regulating bone metabolism and altering migration of cells into givenarea. Non-limiting examples of growth factors include insulin-likegrowth factor-1 (IGF-1), transforming growth factor-β (TGF-β), a bonemorphogenic protein (BMP) and the like.

Non-limiting examples of toxins include the cholera toxin, which alsoserves as an adjuvant.

Non-limiting examples of anti-proliferative agents include an alkylatingagent such as a nitrogen mustard, an ethylenimine and a methylmelamine,an alkyl sulfonate, a nitrosourea, and a triazene; an antimetabolitesuch as a folic acid analog, a pyrimidine analog, and a purine analog; anatural product such as a vinca alkaloid, an epipodophyllotoxin, anantibiotic, an enzyme, a taxane, and a biological response modifier;miscellaneous agents such as a platinum coordination complex, ananthracenedione, an anthracycline, a substituted urea, a methylhydrazine derivative, or an adrenocortical suppressant; or a hormone oran antagonist such as an adrenocorticosteroid, a progestin, an estrogen,an antiestrogen, an androgen, an antiandrogen, or agonadotropin-releasing hormone analog. Specific examples ofchemotherapeutic agents include, for example, a nitrogen mustard, anepipodophyllotoxin, an antibiotic, a platinum coordination complex,bleomycin, doxorubicin, paclitaxel, etoposide, 4-OH cyclophosphamide,and cisplatinum.

The HSP family consists of approximately 25 proteins discerned by theirmolecular weights with highly conserved structures. Almost all humanshave cellular and humoral immune reactions against microbial heat-shockprotein 60 (HSP60). Because a high degree of antigenic homology existsbetween microbial (bacterial and parasitic) and human HSP60, the ‘cost’of immunity to microbes might be the danger of cross-reactivity withhuman HSP60 expressed by the endothelial cells of stressed arteries.Genuine autoimmunity against altered autologous HSP60 might trigger thisprocess also (Wick et al. Atherosclerosis as an autoimmune disease: anupdate. TRENDS in Immunology. 2001; 22(12):665-669). HSP has beenimplicated as a target autoantigen in several experimental autoimmunediseases (arthritis, type I diabetes). Anti-HSP65 as well as anti-HSP60antibodies have been demonstrably associated with atheromatous lesionsin humans. Studies conducted in rabbits and mice show that thegeneration of an HSP65-induced immune response by immunization with therecombinant protein or with an HSP65-rich preparation of Mycobacteriumtuberculosis enhances atherogenesis. As autoimmune processes pointing toHSP65 as a possible antigenic candidate, creating a state ofunresponsiveness by induction of mucosal “tolerization” has beenemployed in order to block these responses, our group reported thatearly atherosclerosis was attenuated in HSP65-fed mice, compared witheither BSA or PBS fed mice (Harats et al. Oral tolerance with heat shockprotein 65 attenuates mycobacterium tuberculosis induced and high fatdiet driven atherosclerosis lesions. J Am Coll Cardiol. 2002;40:1333-1338), this was further supported by Maron who demonstrated thatnasal vaccination with HSP reduces the inflammatory process associatedwith atherosclerosis (Maron et al. Mucosal administration of heat shockprotein-65 decreases atherosclerosis and inflammation in aortic arch oflow density lipoprotein receptor-deficient mice. Circulation. 2002;106:1708-1715).

Beta-2-glycoprotein I (beta2GPI) is a phospholipid binding protein shownto serve as a target for prothrombotic antiphospholipid antibodies. Ithas recently been demonstrated to drive an immune mediated reaction andenhance murine atherosclerosis. β-Antibodies to beta-2-GPI have theability to activate monocytes and endothelial cells and can induce animmune response to beta2GPI in atherosclerosis-prone mice acceleratedatherosclerosis. When beta2GPI-reactive lymph node and spleen cells weretransferred to LDL-receptor-deficient mice they promoted fatty streakformation, proving a direct proatherogenic role for beta2GPI-specificlymphocytes. Inducing immunological tolerance to beta2GPI by prior oralfeeding with the antigen resulted in a significant reduction in theextent of atherosclerotic lesions. Thus, beta2GPI is a candidate playerin the atherosclerotic plaque, and can possibly be employed as animmunomodulator of plaque progression. Oral feeding with of beta2GPIinhibited lymph node cell reactivity to beta2GPI in mice immunizedagainst the human protein. IL-4 and IL-10 production was upregulated inlymph node cells of beta2GPI-tolerant mice immunized against beta2GPI,upon priming with the respective protein. Thus, oral administration ofbeta2GPI is an effective means of suppressing atherogenesis in mice(George et al. Suppression of early atherosclerosis in LDL-receptordeficient mice by oral tolerance with beta2-glycoprotein I. CardiovascRes. 2004; 62(3):603-9).

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include biochemical and immunologicaltechniques. Such techniques are thoroughly explained in the literature.See, for example, “Cell Biology: A Laboratory Handbook”, Volumes I-IIICellis, J. E., ed. (1994); “Current Protocols in Immunology” VolumesI-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic andClinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn.(1994); Mishell and Shiigi (eds), “Selected Methods in CellularImmunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; and “Methods in Enzymology” Vol. 1-317,Academic Press; Marshak et al., all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Materials and General Methods

Animals:

Apo-E knockout mice: Apo-E knockout (Apo-E KO) mice used in ourexperiments are from the atherosclerosis prone strainC57BL/6J-Apoe^(tm1unc). Mice homozygous for the Apoe^(tm1unc) mutationsshow a marked increase in total plasma cholesterol levels which isunaffected by age or sex. Fatty streaks in the proximal aorta are foundat 3 months of age. The lesions increase with age and progress tolesions with less lipid but more elongated cells, typical of a moreadvanced stage of pre-atherosclerotic lesion.

Strain Development: The Apoe^(tm1unc) mutant strain was developed in thelaboratory of Dr. Nobuyo Maeda at University of North Carolina at ChapelHill. The 129-derived E14Tg2a ES cell line was used. The plasmid used isdesignated as pNMC109 and the founder line is T-89. The C57BL/6J strainwas produced by backcrossing the Apoe^(tm1unc) mutation 10 times toC57BL/6J mice (Plump et al., Severe hypercholesterolemia andatherosclerosis in apolipoprotein-E deficient mice created by homologousrecombination in ES cells. Cell 1992; 71: 343-353; Zhang et al.Spontaneous hypercholesterolemia and arterial lesions in mice lackingapolipoprotein E. Science 1992; 258: 468-471).

The mice were maintained at the Sheba Hospital Animal Facility(Tel-Hashomer, Israel) on a 12-hour light/dark cycle, at 22-24° C. andfed a normal fat diet of laboratory chow (Purina Rodent Laboratory ChowNo. 5001) containing 0.027% cholesterol, approximately 4.5% total fat,and water, ad libitum.

LDL-RD mice: LDL-RD Mice [LDLr<mlHer>LDL−/−(C57B/6 50% JSL 25% I12925%)], 8-12 weeks old, were supplied by the Hadassah Hospital AnimalFacility (Hadassah Hospital, Israel).

Lewis rats: Male Lewis rats, aged 9-11 weeks, were supplied by Harlanlaboratories (ISRAEL)

Immunization:

I. Intraperitoneal immunization with ALLE: The phospholipid ether analog(ALLE D+L) was coupled to purified protein derivative from tuberculin(PPD). The stock solution of ALLE (D+L) was dissolved in ethanol (99mg/ml). 5 mg ALLE (D+L), (50.5 μl from stock solution) was diluted to 5mg/ml with 0.25M phosphate buffer, pH 7.2, by stirring at 0° C. (in anice bath). 1.5 mg of D- and L-ALLE in 300 μl of phosphate buffer wereadded to 0.6 mg PPD dissolved in 300 μl of phosphate buffer.1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimid-HCl (5 mg; Sigma, St.Louis, Mo.) dissolved in 50 μl of water was added by stirring at 4° C.for 20 minutes. The remaining active sites were blocked with 100 μl of1M glycine. Coupled compounds were dialyzed against phosphate-bufferedsaline (PBS), adjusted to 3 ml with PBS and stored at 4° C. Immunizationwith 0.3 ml (150 μg) antigen per mouse was performed intraperitoneally 4times every 2 weeks.

II. Subcutaneous immunization with Human oxidized LDL: Human oxidizedLDL was prepared from human plasma pool (d-1.019 to 1.063 gram/ml byultracentrifugation) and was Cu-oxidized overnight (by adding 15 μl 1 mMCuSO₄ to each ml of LDL previously diluted to 1 mg/ml concentration).The oxidized LDL was dialyzed against PBS and filtrated. Forimmunization, oxidized LDL was dissolved in PBS and mixed with equalvolumes of Freund's incomplete adjuvant. Immunizations were performed bysingle subcutaneous injection of 50 μg antigen/mouse in 0.2 ml volume.One to three days following the last oral administration the micereceived one immunization, and were sacrificed 7-10 days postimmunization.

Cholesterol Level Determination: At the completion of the experiment,1-1.5 ml of blood was obtained by cardiac puncture, 1000 U/ml heparinwas added to each sample and total plasma cholesterol levels weredetermined using an automated enzymatic technique (Boehringer Mannheim,Germany).

FPLC Analysis: Fast Protein Liquid Chromatography analysis ofcholesterol and lipid content of lipoproteins was performed usingSuperose 6 HR 10/30 column (Amersham Pharmacia Biotech, Inc, Peapack,N.J.) on a FPLC system (Pharmacia LKB. FRAC-200, Pharmacia, Peapack,N.J.). A minimum sample volume of 300 μl (blood pooled from 3 mice wasdiluted 1:2 and filtered before loading) was required in the samplingvial for the automatic sampler to completely fill the 200 μl sampleloop. Fractions 10-40 were collected, each fraction contained 0.5 ml. A250 μl sample from each fraction was mixed with freshly preparedcholesterol reagent or triglyceride reagent respectively, incubated for5 minutes at 37° C. and assayed spectrophotometrically at 500 nm.

Assessment of Atherosclerosis: Quantification of atherosclerotic fattystreak lesions was done by calculating the lesion size in the aorticsinus as previously described (Paigen et al. Quantitative assessment ofatherosclerotic lesions in mice. Atherosclerosis 1987; 68: 231-140) andby calculating the lesion size in the aorta. Briefly, after perfusionwith saline Tris EDTA, the heart and the aorta were removed from theanimals and the peripheral fat cleaned carefully. The upper section ofthe heart was embedded in OCT medium (10.24% w/w polyvinyl alcohol;4.26% w/w polyethylene glycol; 85.50% w/w nonreactive ingredients) andfrozen. Every other section (10 μm thick) throughout the aortic sinus(400 μm) was taken for analysis. The distal portion of the aortic sinuswas recognized by the three valve cusps that are the junctions of theaorta to the heart. Sections were evaluated for fatty streak lesionsafter staining with oil-red 0. Lesion areas per section were scored on agrid (Rubin et al. Inhibition of early atherogenesis in transgenic miceby human apoplipoprotein A-I. Nature 1991; 353: 265-267) by an observercounting unidentified, numbered specimens. The aorta was dissected fromthe heart and surrounding adventitious tissue was removed. Fixation ofthe aorta and Sudan staining of the vessels were performed as previouslydescribed (Bauman and Mangold, J. Org. Chem. 31: 498, 1966).

Plasma Measurements and Quantification of Atherosclerotic Lesions:Plasma total cholesterol and total triglyceride levels were measured byCOBAS MIRA. Hearts were collected upon sacrifice and the aortic rootcryosections were stained using Oil-Red-O staining. Atheroscleroticlesion area was evaluated by computer analysis (Image Pro Plus) andsupported by microscope evaluation as well.

Proliferation assays: Mice were fed with ALLE, POVPC or PBS as describedfor assessment of atherosclerosis, and then immunized one day followingthe last feeding with oxidized LDL prepared from purified human LDL asdescribed above.

Proliferation was assayed eight days after immunization with theoxidized LDL as follows: Spleens or lymph nodes were prepared by meshingthe tissues on 100 mesh screens. (Lymph nodes where immunization wasperformed, and spleens where no immunization performed). Red blood cellswere lysed with cold sterile double distilled water (6 ml) for 30seconds and 2 ml of NaCl 3.5% were added. Incomplete medium was added(10 ml), cells were centrifuge for 7 minutes at 1,700 rpm, resuspendedin RPMI medium and counted in a haemocytometer at 1:20 dilution (10 μlcells+190 μl Trypan Blue). Proliferation was measured by theincorporation of [³H]-Thymidine into DNA in triplicate samples of 100 μlof the packed cells (2.5×10⁶ cells/ml) in a 96 well microtiter plate.Triplicate samples of oxidized LDL (0-10 μg/ml, 100 μl/well) were added,cells incubated for 72 hours (37° C., 5% CO₂ and about 98% humidity) and10 μl ³-[H]-Thymidine (0.5 μCi/well) were added. After an additional dayof incubation the cells were harvested and transferred to glass fiberfilters using a cell harvester (Brandel) and counted using β-counter(Lumitron). For assay of cytokines the supernatant was collected withoutadding ³-[H]-Thymidine and assayed by ELISA.

A separate group of mice were fed with ALLE or PBS and immunized withoxidized LDL as described above, one day following the last fed dose.Draining inguinal lymph nodes (taken 8 days after immunization) werecollected from 3 mice from each of the groups, for the proliferationstudies. 1×10⁶ cells per ml were incubated in triplicates for 72 hoursin 0.2 ml of culture medium in microtiter wells in the presence 10 μg/mloxidized LDL. Proliferation was measured by the incorporation of[³H]-thymidine into DNA during the final 12 hours of incubation. Theresults are expressed as the stimulation index (S.I.):the ratio of themean radioactivity (cpm) of the antigen to the mean background (cpm)obtained in the absence of the antigen. Standard deviation was always<10% of the mean cpm.

Inflammatory Markers Evaluation in Serum: Serum was separated bycentrifuge and stored at −70° C. Analysis of inflammatory markers wasperformed by ELISA (IL-10; R&D and SAA; BIOSOURCE).

RT-PCR analysis: Aortas, spleens and small intestine were removed out oftreated and untreated mice (in a sterile manner) and freezed in liquidnitrogen. The organs were mashed on screen cup and the RNA productionwas performed using Rneasy kit (QIAGEN). RNA samples were examined inspectrophotometer and normalized relative to β-actin. Reversetranscription of RNA to cDNA and PCR with primers was performed with“Titan one tube RT-PCR kit” (ROCHE). Results were detected on 1% agarosegel and were documented on film.

Statistical Analysis: A one-way ANOVA test was used to compareindependent values. p<0.05 was accepted as statistically significant.

Example I Synthesis of the immunomodulizing antigens 2,5′-AldehydeLecithin Ether (ALLE) and POVPC

Synthesis of 2,5′-Aldehyde Lechitin Ether (ALLE)

2,5′-Aldehyde Lecithin Ether (ALLE) was synthesized according to amodification of general methods for synthesis of ether analogs oflecithins communicated by Eibl H., et al. Ann. Chem. 709:226-230,(1967), W. J. Baumann and H. K. Mangold, J. Org. Chem. 31,498 (1996), E.Baer and Buchnea JBC. 230,447 (1958), Halperin G et al Methods inEnzymology 129, 838-846 (1986). The following protocol refers tocompounds and processes depicted in 2-D form in FIG. 1.

Hexadecyl-glycerol ether: D-Acetone glycerol (4 grams) for synthesis ofL-ALLE or L-Acetone glycerol for synthesis of D-ALLE, powdered potassiumhydroxide (approximately 10 grams) and hexadecyl bromide (9.3 grams) inbenzene (100 ml) were stirred and refluxed for 5 hours, while removingthe water formed by azeotropic distillation (compare W. J. Baumann andH. K. Mangold, J. Org. Chem. 29: 3055, 1964 and F. Paltauf, Monatsh.99:1277, 1968). The volume of the solvent was gradually reduced to about20 ml, and the resulting mixture was cooled to room temperature anddissolved in ether (100 ml). The resulting solution was washed withwater (2×50 ml), and the solvent was removed under reduced pressure. A100 ml mixture of 90:10:5 methanol:water:concentrated hydrochloric acidwas added to the residue and the mixture was refluxed for 10 minutes.The product was extracted with ether (200 ml) and was washedconsecutively with water (50 ml), 10% sodium hydroxide (20 ml) and againwith water (volumes of 20 ml) until neutral. The solvent was removedunder reduced pressure and the product (8.8 grams) was crystallized fromhexane to give 7.4 grams of pure 1-hexadecyl-glyceryl ether (compound I,FIG. 1) for synthesis of D-ALLE or 3-hexadecyl-glyceryl ether forsynthesis of L-ALLE.

5-Hexenyl-methane sulfonate: A mixture of 5-hexene-1-ol (12 ml) and drypyridine (25 ml) was cooled to between −4° C. and −10° C. in an ice-saltbath. Methanesulfonyl chloride (10 ml) was added dropwise during aperiod of 60 minutes, and the mixture was kept at 4° C. for 48 hours.Ice (20 grams) was added, the mixture was allowed to stand for 30minutes, and the product was extracted with ether (200 ml). The organicphase was washed with water (20 ml), 10% hydrochloric acid, 10% sodiumbicarbonate (20 ml) and again with water (20 ml). The solvent wasevaporated and the crude product was chromatographed on silica gel 60(100 grams) using a mixture of 80:20 CHCl₃:EtOAc as eluent, to give 14grams of 5-hexenyl-methane sulfonate.

J-Hexadecyloxy-3-trityloxy-2-propanol (for D-ALLE) or3-Hexadecyloxy-1-trityloxy-2-propanol (for L-ALLE) (compound II):1-Hexadecyloxy-glycerol (for D-ALLE) or 3-Hexadecyloxy-glycerol (forL-ALLE) (7.9 grams), triphenylchloromethane (8.4 grams) and dry pyridine(40 ml) were heated at 100° C. for 12 hours. After cooling, 300 ml ofether and 150 ml of ice-cold water were added, and the reaction mixturewas transferred to a separatory funnel. The organic phase was washedconsecutively with 50 ml of ice water, 1% potassium carbonate solution(until basic) and 50 ml of water, then dried over anhydrous sodiumsulfate. The solvent was evaporated, the residue was dissolved in 150 mlof warm petroleum ether and the resulting solution was cooled at 4° C.over night. After filtration of the precipitate, the filtrate wasevaporated and the residue was recrystallized from 20 ml of ethylacetate at −30° C., yielding 8.2 grams of1-Hexadecyloxy-3-trityloxy-2-propanol (for D-ALLE) or3-hexadecyloxy-1-trityloxy-2-propanol (for L-ALLE) (compound II, FIG.1), melting point 49° C.

1-Hexadecyl-2-(5′-hexenyl)-glyceryl ether (for D-ALLE) or3-hexadecyl-2-(5′-hexenyl)-glyceryl ether (for L-ALLE) (compound IV):1-Hexadecyloxy-3-trityloxy-2-propanol (for D-ALLE) or3-Hexadecyloxy-1-trityloxy-2-propanol (for L-ALLE) (compound II, FIG. 1)(5.5 grams) was etherified with 5-hexenyl-methanesulfonate, usingpowdered potassium hydroxide in benzene solution as described above. Thecrude product 1-Hexadecyloxy-2-(5′-hexenyloxy)-sn-3-trityloxy-propane(for D-ALLE) or 3-Hexadecyloxy-2-(5′-hexenyloxy)-sn-3-trityloxy-propane(for L-ALLE) (compound III, FIG. 1) was dissolved in 100 ml of 90:10:5methanol:water:concentrated hydrochloric acid and the mixture wasrefluxed for 6 hours. The product was extracted with ether, washed withwater and the solvent was removed. The residue was dissolved inpetroleum ether (100 ml) and was kept in 4° C. for overnight, causingmost of the triphenyl carbinol to be deposited. After filtration andremoval of the solvent from the filtrate the crude product waschromatographed on silica gel 60 (40 grams), using a mixture of 1:1chloroform:petroleum ether as eluent, to give 1.8 grams of pure1-hexadecyl-2-(5′-hexenyl)-glyceryl ether (for D-ALLE) or3-hexadecyl-2-(5′-hexenyl)-glyceryl ether (for L-ALLE) (compound IV,FIG. 1).

1-Hexadecyl-2-(5′-hexenyl)-sn-glycero-3-phosphocholine (for D-ALLE) or3-Hexadecyl-2-(5′-hexenyl)-sn-glycero-1-phosphocholine (for L-ALLE)(compound V): The following procedure is a modification of the methodcommunicated by Eibl H., et al. Ann. Chem. 709:226-230, 1967.

A solution of 1-hexadecyl-2-hexenyl-glyceryl ether (for D-ALLE) or3-hexadecyl-2-hexenyl-glyceryl ether (for L-ALLE) (compound IV, FIG. 1)(2 grams) in dry chloroform (15 ml) was added dropwise into a stirred,cooled solution (−4° C. to −10° C.) of dry triethylamine (3 ml) and2-bromoethyl dichlorophosphate (1.25 ml, prepared as describedhereinbelow) in dry chloroform (15 ml), during a period of 1 hour. Themixture was kept at room temperature for 6 hours and then heated to 40°C. for 12 hours. The resulting dark brown solution was cooled to 0° C.and 0.1M potassium chloride (15 ml) was added. The mixture was allowedto reach room temperature and was stirred for 60 minutes. Methanol (25ml) and chloroform (50 ml) were added and the organic phase was washedwith 0.1M hydrochloric acid (20 ml) and water (20 ml). The solvent wasevaporated and the crude product was dissolved in methanol (15 ml), thesolution was transferred to a culture tube and was cooled in an ice-saltbath. Cold trimethylamine (3 ml, −20° C.) was added and the tube wassealed. The mixture was heated to 55° C. for 12 hours, cooled to roomtemperature and the solvent evaporated using a stream of nitrogen. Theresidue was extracted with a mixture of 2:1 chloroform:methanol (25 ml)and washed with 1M potassium carbonate (10 ml) and water (2×10 ml). Thesolvent was removed under reduced pressure and the crude products werechromatographed on silica gel 60 (20 grams), using a mixture of 60:40chloroform:methanol, to give 1.5 grams of1-hexadecyl-2-(5′-hexenyl)-sn-glycero-3-phosphocholine (for D-ALLE) or3-hexadecyl-2-(5′-hexenyl)-sn-glycero-1-phosphocholine (for L-ALLE)(compound V, FIG. 1). The structure of compound V was confirmed by NMRand mass spectrometry.

1-Hexadecyl-2-(5′-oxo-pentanyl)-sn-glycero-3-phosphocholine (for D-ALLE)or 3-Hexadecyl-2-(5′-oxo-pentanyl)-sn-glycero-1-phosphocholine (forL-ALLE) (compound VI)

A solution of Compound V (0.5 gram) in formic acid (15 ml) and 30%hydrogen peroxide (3.5 ml) was stirred at room temperature over night.The reaction mixture was diluted with water (50 ml), and extracted witha mixture of 2:1 chloroform:methanol (5×50 ml). The solvent wasevaporated under reduced pressure and the residue was mixed withmethanol (10 ml) and water (4 ml), then stirred at room temperature for60 minutes. 80% phosphoric acid (2 ml) and potassium meta periodate (0.8gram) were then added. The mixture was kept at room temperatureovernight, diluted with water (50 ml) and extracted with 2:1chloroform:methanol (50 ml). The organic phase was washed with 10%sodium bisulfite (10 ml) and water (10 ml). The solvent was removedunder reduced pressure and the crude product was chromatographed onsilica gel 60 (10 grams), using a mixture of 1:1 chloroform:methanol aseluent, to give 249 mg of1-hexadecyl-2-(5′-oxo-pentanyl)-sn-glycero-3-phosphocholine (for D-ALLE)or 3-hexadecyl-2-(5′-oxo-pentanyl)-sn-glycero-1-phosphocholine (forL-ALLE) (compound VI, FIG. 1), exhibiting an R_(f) of 0.15 (TLC system,60:40:8 chloroform:methanol:water) and a positive reaction withdinitrophenylhydrazine. The chemical structure of Compound VI wasconfirmed by NMR and mass spectrometry.

In an alternative process, the ethylenic group was converted to analdehyde group by ozonization and catalytic hydrogenation with palladiumcalcium carbonate.

Preparation of 2-bromoethyl dichlorophosphate: 2-Bromoethyldichlorophosphate was prepared by dropwise addition of freshly distilled2-bromoethanol (0.5 M, prepared as described in Gilman Org. Synth.12:117, 1926) to an ice-cooled solution of freshly distilled phosphorousoxychloride (0.5 M) in dry chloroform, during a one hour period,followed by 5 hours reflux and vacuum distillation (bp 66-68° C. at0.4-0.5 mm Hg). The reagent was stored (−20° C.) under nitrogen in smallsealed ampoules prior to use (Hansen W. H et al. Lipids 17(6):453-459,1982).

Synthesis of1-Hexadecyl-2-(5′-carboxy-butyl)-sn-glycero-3-phosphocholine (CI-201)

1-Hexadecyl-2-(5′-oxo-pentanyl)-sn-glycero-3-phosphocholine (CompoundVI, prepared as described above), 0.55 grams (0.001 mol), was dissolvedin t-BuOH (30 ml). A solution of NaClO₂ (0.9 gram, 0.01 mol) and NaH₂PO₄(0.96 gram, 0.07 mol) in 25 ml water was added dropwise during a periodof 30 minutes and the mixture was stirred at room temperature for 3hours. The reaction mixture was acidified to pH=3 with concentratedhydrochloric acid and extracted with a mixture of 2:1chlroform:methanol. The organic phase was separated and the solvent wasevaporated. The residue was purified by chromatography over silica gelusing a mixture of chloroform:methanol:water (70:27:3), to give1-hexadecyl-2-(5′-carboxy-butyl)-sn-glycero-3-phosphocholine (0.42 gram,72% yield). NMR and mass spectrometry confirmed the chemical structure(Compound VII, FIG. 10).

Synthesis of1-Hexadecyl-2-(5′,5′-dimethoxy-pentyloxy)-sn-glycero-3-phosphocholine

1-Hexadecyl-2-(5′-oxo-pentanyl)-sn-glycero-3-phosphocholine (compoundVI, prepared as described above), 0.50 gram (0.89 mmol), was dissolvedin formic acid (15 ml) and hydrogen peroxide 30% (3.5 ml) was added. Thereaction mixture was stirred overnight at room temperature. Afteraddition of water (50 ml) the product was extracted with a mixture of2:1 chloroform:methanol (2×50 ml). The organic phase was washed withaqueous 10% sodium bicarbonate (10 ml) and water (10 ml) and the solventwas removed under reduced pressure. The residue (0.4 gram) was dissolvedin methanol (10 ml), aqueous 10% sodium hydroxide (4 ml) was then addedand the reaction mixture was stirred at room temperature for 1 hour. 80%Phosphoric acid (2 ml) and potassium meta periodate (0.8 gram) werethereafter added and stirring was continued for over night. A mixture of2:1 chloroform:methanol (50 ml) was then added and the organic phase waswashed with aqueous 10% sodium bisulfite (10 ml) and water (10 ml), andthe solvent was removed under vacuum. The residue (0.3 gram) waspurified by chromatography over silica gel (10 grams) using a mixture ofchloroform:methanol (60:40 to 40:60) as graduated eluent, to give1-hexadecyl-2-(5′,5′-dimethoxy-pentyloxy)-sn-glycero-3-phosphocholine(0.25 gram, 46% yield). NMR and mass spectrometry confirmed the chemicalstructure (Compound VIIIa, FIG. 10).

Synthesis of1-Hexadecyl-2-(5′,5′-diethoxypentyloxy)-sn-glycero-3-phosphocholine

Crude 1-Hexadecyl-2-(5′-oxo-pentanyl)-sn-glycero-3-phosphocholine(compound VI, prepared as described above), 50 mg (0.088 mmol), wasdissolved in ethanol (10 ml), under a nitrogen atmosphere. Triethylorthoformate (0.053 ml, 0.0476 gram, 0.32 mmol) and 3 drops of conc.sulfuric acid were added and the reaction mixture was stirred overnightat room temperature. Dichloromethane (75 ml) was then added and thereaction mixture was transferred to a separatory funnel, washedsuccessively with water (75 ml), aqueous 2.5% sodium bicarbonatesolution (75 ml) and water (75 ml), and was dried over anhydrous sodiumsulfate. After filtration, the solvent was removed under vacuum, to give50 mg of crude1-hexadecyl-2-(5′,5′-diethoxypentyloxy)-sn-glycero-3-phosphocholine. Thestructure was confirmed by CMR and MS spectroscopy (Compound VIIIb, FIG.10).

Synthesis of1-Hexadecanoyl-2-(5′-oxo-valeroyl)-sn-3-glycerophosphocholine(POVPC)

A mixture of 1-hexadecanoyl-sn-3-glycerophosphocholine (compound I, FIG.2) (3 grams), 5-hexenoic acid (1.2 ml), 1,3-dicyclohexylcarbodiamide(DCC, 4.05 grams) and N,N-dimethylaminopyridine (DMP, 1.6 grams) indichloromethane (100 ml, freshly distilled from phosphorus pentoxide)was thoroughly stirred for 4 days at room temperature. The mixture wasthen chromatographed on silica gel 60 (40 grams) and the product,1-hexadecanoyl-2-(5′-hexenoyl)-sn-3-glycerophosphocholine (2.8 grams,compound II, FIG. 2) was eluted with a mixture of 25:75chloroform:methanol. The eluent was dissolved in 30% hydrogenperoxide:formic acid (4:15) and the solution was stirred overnight atroom temperature. Water (50 ml) were added, the product was extractedwith 2:1 chloroform:methanol (100 ml) and the organic phase was washedwith water. The solvent was evaporated under reduced pressure, theresidue was dissolved in methanol (15 ml) and 10% ammonia solution (5ml) and the resulting solution was stirred at room temperature for 6hours. The crude1-hexadecanoyl-2-(5′,6′-dihydroxy)-hexanoyl-sn-3-glycerophosphocholine(compound III, FIG. 2) (structure confirmed by NMR and massspectrometry) was further reacted without purification. 80% phosphoricacid (3 ml) and sodium metaperiodate (1 gram) were added to the solutionand the mixture was stirred at room temperature for overnight, and wasthereafter extracted with a mixture of 2:1 chloroform:methanol. Theproduct was purified by chromatography on silica gel 60 (20 grams),using a mixture of 25:75 chloroform:methanol as eluent. 850 mg of1-hexadecanoyl-2-(5′-oxo-valeroyl)-sn-3-glycerophosphocholine (POVPC,compound IV, FIG. 2) were obtained, exhibiting chromatographic mobilityof lecithin on TLC, and positive dinitrophenyl hydrazine reaction. Thestructure was assessed by NMR and mass spectrometry.

Alternatively: the ethylenic group was converted to an aldehyde byozonization and catalytic hydrogenation with palladium calciumcarbonate.

Example II Immunization Against L-ALLE+D-ALLE Specifically InhibitsAtherogenesis in Genetically Disposed (Apo-E KO) Mice

The present inventors have demonstrated that immunization with thestable, etherified synthetic LDL component ALLE can reduce the extent ofatherosclerotic plaque formation in susceptible mice. 19 female 5-7weeks old Apo E/C 57 mice were divided into 3 groups. In group A (n=6)the mice were immunized intraperitoneally, as described in Materials andMethods section above, with 150 μg/mouse L-ALLE+D-ALLE once every 2weeks (0.3 ml/mouse) X4. In group B (n=6) the mice were immunized withtuberculin toxin Purified Protein Derivative (PPD) once every 2 weeks(0.3 ml/mouse). In group C (n=7) the mice received no immunization. Micefrom all three groups were bled prior to immunization (Time 0), and oneweek after the second immunization for determination of anti-ox LDLantibodies, anti-ALLE antibodies and lipid profile. Atherosclerosisassessment was performed as described above, 4.5 weeks post 4^(th)immunization. The mice from all groups were weighed at 2 week intervalsthroughout the experiment. All mice were fed normal chow-diet containing4.5% fat by weight (0.02% cholesterol) and water ad libitum.

TABLE I Immunization of Apo-E KO mice with ALLE inhibits atherogenesis150 μg/Mouse L-ALLE + Control D-ALLE without immunization PPDimmunization Groups N = 6 N = 5 N = 7 Statistics Time 0 Weight 17.3 ±0.5 17.3 ± 0.7 17.8 ± 0.4 P = 0.780 Chol 435 ± 47 436 ± 49 413 ± 44 P =0.919 TG 118 ± 9  112 ± 10 120 ± 14 P = 0.865 End Weight 20.5 ± 0.5 21.6± 0.2 20.3 ± 0.5 P = 0.123 Chol 299 ± 18 294 ± 15 3044 ± 22 P = 0.937 TG57 ± 3 53 ± 4 66 ± 4 P = 0.075 Lesion 101000 ± 8276  179500 ± 13449210833 ± 26714 P = 0.005 size (μm²) TGF-β 12032 ± 2308 13963 ± 944 12825 ± 2340 P = 0.831 pmol/ml Note: “Weight” is weight in grams; “Chol”is plasma cholesterol and “TG” is plasma triglycerides, expressed inmg/dL.

As can be seen in FIG. 3, the results depicted in Table I demonstratethe significant reduction in atheromatous lesions measured in the hearttissues of the ALLE immunized mice, compared to both PPD and unimmunizedcontrol mice. No significant effect is apparent on other parametersmeasured, such as weight gain, triglyceride or cholesterol plasmalevels, or immune competence, as measured by the levels of theimmunosuppressive cytokine TGF-β. Thus, immunization with the syntheticoxidized LDL component ALLE (a mixture of racemic forms D- and L-)confers significant (>50%) protection from atherosclerotic lesionformation in these genetically susceptible Apo-E KO mice. A significantbut less dramatic reduction in plaquing was observed in mice immunizedwith PPD.

Example III Inhibition of Atherogenesis in Genetically Predisposed(Apo-E KO) Mice by Oral Administration of L-ALLE and D-ALLE

Intraperitoneal immunization with the ester analogs of plaque lesioncomponents was effective in inhibiting atherogenesis in Apo-E KO mice(FIG. 1). Thus, the ability of oral administration of L- and D-ALLE tosuppress atherogenesis was investigated. 34 male 8-10 week old Apo-E KOmice were divided into three groups. In group A (n=11), mice were orallyadministered by gavage of L-ALLE+D-ALLE suspended in PBS 5% ethanol (1mg/mouse) for 5 days every other day. In group B (n=11) mice were fedwith 10 μg/mouse L-ALLE+D-ALLE suspended in PBS 5% ethanol for 5 daysevery other day. (0.2 ml/mouse). Mice in group C (n=12) received PBS(containing the same volume of ethanol as in groups A+B). Mice were bledprior to feeding (Time 0) and at the conclusion of the experiment (End)for determination of lipid profile. Atherosclerosis was assessed in theaorta sinus, as described above, 8 weeks after the last feeding. Micewere weighed every 2 weeks during the experiment. All mice were fednormal chow-diet containing 4.5% fat by weight (0.02% cholesterol) andwater ad libitum.

TABLE 2 Inhibition of atherogenesis in Apo-E KO out mice by oraladministration of L-ALLE and D-ALLE PBS 1 mg ALLE 10 μg ALLE Groups N =12 N = 11 N = 11 Statistics Time 0 Weight 20.7 ± 0.6 21.5 ± 0.8 21.1 ±0.8 P = 0.794 Chol 373 ± 25 379 ± 23 378 ± 31 P = 0.983 TG 128 98 90 P =0.829 End Weight 27.3 ± 0.4 27.4 ± 0.5 24.1 ± 0.8 P < 0.001 Chol 303 ±17 249 ± 24 321 ± 15 P = 0.031 TG 81 ± 4 78 ± 8 93 ± 6 P = 0.146 Lesionsize 176000 ± 13735  85278 ± 11633 103889 ± 14320 P < 0.001 (μm²) TGF-β14696 ± 1352 13388 ± 1489 18010 ± 1373 P = 0.07 pmol/ml Note: “Weight”is weight in grams; “Chol” is serum cholesterol and “TG” is serumtriglycerides, expressed in mg/dL.

As can be seen from FIG. 4, the results depicted in Table 2 demonstratea striking attenuation of atherosclerotic progression measured in thetissues of mice fed low doses (10 μg-1 mg/mouse) of ALLE, compared tounexposed control mice. No significant effect is apparent on otherparameters measured, such as weight gain, triglyceride or cholesterolblood levels, or immune competence, as measured by the levels of theimmunosuppressive cytokine TGF-β. Thus, oral administration of thesynthetic oxidized LDL component ALLE provides for significant (>50%)protection from atherosclerosis in these genetically susceptible Apo-EKO mice, similar to the protection achieved with peritoneal immunization(see FIG. 1).

Example IV Inhibition of Atherogenesis in Genetically Predisposed (Apo-EKO) Mice by Induction of Oral- and Nasal-Mediated Immunomodulation withL-ALLE

Mechanisms of mucosal-mediated immunomodulation are active in the nasalmucosa as well as the gut. Thus, nasal exposure and oral exposure to L-and D-ALLE were compared for their effectiveness in suppressingatherogenesis in Apo-E KO mice. 34 male 7-10 weeks old Apo-E KO micewere divided into 3 groups. In group A (n=11) mice were orallyadministered with L-ALLE suspended in PBS 5% ethanol (1 mg/mouse/0.2 ml)for 5 days every other day. In group B (n=11) mice were nasallyadministered as described above in Materials and Methods with 10μg/mouse/10 μL-ALLE suspended in PBS every other day for 3 days. Mice ingroup C (n=12) received PBS administered orally and nasally (containingthe same volume of ethanol as in groups A+B). Mice were bled prior tofeeding (Time 0) and at the conclusion of the experiment (End) fordetermination of lipid profile. Atherosclerosis was assessed in theaorta sinus as described above, 8 weeks after the last feeding. Micewere weighed every 2 weeks during the experiment. All mice were fednormal chow-diet containing 4.5% fat by weight (0.02% cholesterol) andwater ad libitum.

TABLE 3 The effect of oral and nasal administration of L-ALLE onmetabolic parameters and atherogenesis in Apo-E KO mice 1 mg 10 μg ALLEALLE PBS Oral Nasal Oral/nasal Groups (N = 11) (N = 11) (N = 12)Statistics Time 0 Weight 21.1 ± 0.8 21.1 ± 0.7 22.1 ± 0.9 P = 0.624 Chol362 ± 27 353 ± 31 351 ± 27 P = 0.952 TG 144 143 138 P = 0.977 End Weight23.3 ± 1.1 24.2 ± 0.2 24.0 ± 0.5 P = 0.558 Chol 418 ± 43 328 ± 18 343 ±25 P = 0.084 TG 82 ± 7 74 ± 6 79 ± 5 P = 0.630 Lesion size  76944 ±17072  82708 ± 10986 135455 ± 12472 P = 0.007 (μm²) Note: “Weight” isweight in grams; “Chol” is plasma cholesterol and “TG” is plasmatriglycerides, expressed in mg/dL.

As can be seen from FIG. 5, the results depicted in Table 3 demonstrateeffective inhibition of atherogenesis measured in the tissues of micereceiving nasal exposure to low doses (10 μg/mouse) of ALLE, compared tounexposed control mice. Nasal administration, like oral administration,had no significant effect on other parameters measured, such as weightgain, triglyceride or cholesterol plasma levels. Thus, the syntheticoxidized LDL component ALLE provides for significant (approximately 50%)protection from atherogenesis in these genetically susceptible Apo-E KOmice, by both oral and nasal administrations.

Example V Suppression of Specific Anti-oxLDL Immune Reactivity inGenetically Predisposed (Apo-E KO) Mice by Oral Administration of L-ALLEor POVPC

Immunomodulation induced by mucosal exposure to oxidized analogs of LDLmay be mediated by suppression of specific immune responses to theplaque-related antigens. POVPC(1-Hexadecanoyl-2-(5′-oxo-valeroyl)-sn-glycerophosphocholine) is anon-ether oxidized LDL analog, which, unlike ALLE is susceptible tobreakdown in the liver and gut. Lymphocyte proliferation in response tooral exposure to both POVPC and the more stable analog ALLE was measuredin Apo-E KO mice. 8 male, 6 week old Apo-E KO mice were divided into 3groups. In group A (n=2) mice were fed with 1 mg/mouse L-ALLE suspendedin 0.2 ml PBS, administered by gavage, as described above, every otherday for 5 days. In group B (n=3) mice were fed with 1 mg/mouse POVPCsuspended in 0.2 ml PBS, administered per os as described above, everyother day for 5 days. The mice in group C (n=3, control) were fed with200 μl PBS every other day for 5 days. Immune reactivity was stimulatedby immunization with Human oxidized LDL as described above in theMaterials and Methods section, one day after the last feeding. One weekafter the immunization lymph nodes were collected for assay ofproliferation. All mice were fed normal chow-diet containing 4.5% fat byweight (0.02% cholesterol) and water ad libitum.

TABLE 4 Oral pretreatment with synthetic oxidized LDL (ALLE and POVPC)suppresses immune response to Human ox-LDL in Apo-E KO mice StimulationIndex (SI) PBS POVPC L-ALLE statistics 33.1 ± 6.1 10.6 ± 2.3 7.3 ± 2.3 P< 0.01 N = 3 N = 3 N = 2 −68% −78%

As can be seen from FIG. 6, the results depicted in Table 4 demonstratesignificant suppression of immune reactivity to Human oxidized-LDLantigen, measured by inhibition of proliferation in the lymph nodes ofApo-E KO mice. Lymphocytes from mice receiving oral exposure toatherogenesis-inhibiting doses (1 mg/mouse) of ALLE or POVPC showed areduced stimulation index following immunization with ox-LDL, ascompared to control (PBS) mice. Since induction of oral-mediated, likenasal-mediated, immunomodulation had no significant effect on otherparameters measured, such as weight gain, triglyceride or cholesterolplasma levels, or immune competence (see Tables 1, 2 and 3), theseresults indicate a specific suppression of anti-ox-LDL immunereactivity. Thus, oral administration of the synthetic oxidized LDLcomponent L-ALLE is an effective method of attenuating the cellularimmune response to immunogenic and atherogenic plaque components inthese genetically susceptible Apo-E KO mice. FIG. 4 also demonstrates asimilar, if less effective inhibition of proliferation with oraladministration of the less stable synthetic oxidized LDL componentPOVPC.

Example VI Inhibition of Atherogenesis in Genetically Predisposed (Apo-EKO) Mice by Oral Administration of D- and L-Isomers of ALLE, and POVPC

Since feeding of ALLE and POVPC was shown to inhibit early atherogenesisand immune reactivity to plaque-related Human LDL antigen, the abilityof both D- and L-isomers of the ether LDL analog, and the non-etheranalog POVPC to suppress the progression of atherogenesis in older micewas compared. Their effect on the triglyceride and cholesterol fractionsof VLDL was also monitored by FPLC. 57 male, 24.5 week old Apo-E KO micewere divided into 5 groups. In group A (n=11) mice were fed with 1mg/mouse L-ALLE suspended in 0.2 ml PBS, administered by gavage, asdescribed above, every other day for 5 days. In group B (n=9) mice werefed with 1 mg/mouse D-ALLE suspended in 0.2 ml PBS, administered per os,as described above, every other day for 5 days. In group C (n=10) micewere fed with 1 mg/mouse POVPC suspended in 0.2 ml PBS, administered bygavage, as described above, every other day for 5 days. Control group D(n=10) received oral administration of PBS (containing the same volumeof ethanol as in groups A, B, C). Base line group was sacrificed ontime=0. Oral administration of the tested antigens took place every 4weeks (5 oral feedings; every other day) starting at 24.5 weeks age,during 12 weeks (3 sets of feedings).

Mice were bled prior to feeding (Time 0), after the 2^(nd) set offeeding and at the conclusion of the experiment (End) for determinationof lipid profile, lipid fractionation and plasma collection.Atherosclerosis was assessed as described above in the aorta sinus andaorta. Spleens were collected for proliferation assay 12 weeks after thefirst feeding. Weight was recorded every 2 weeks throughout theexperiment. All mice were fed normal chow-diet containing 4.5% fat byweight (0.02% cholesterol) and water ad libitum.

TABLE 5 Inhibition of atherogenesis in Apo-E KO mice by oraladministration of L-ALLE, D-ALLE and POVPC 1 mg 1 mg 1 mg Base line TimeParameter PBS L-ALLE D-ALLE POVPC (t = 0) point tested (n = 10) (n = 11)(n = 9) (n = 10) (n = 8) statistics 0 Weight 28.1 ± 0.5   29 ± 0.6 29.8± 0.7 29.6 ± 0.7 29.8 ± 1.1 P = 0.445 Cholesterol 413 ± 27 413 ± 23 409± 28 401 ± 21 393 ± 16 P = 0.976 Triglyceride 67 ± 5 63 ± 8 63 ± 4 67 ±7 71 ± 8 P = 0.946 End Weight 28.5 ± 0.6 29.7 ± 0.5 30.4 ± 0.8 29.9 ±0.5 — P = 0.177 Cholesterol 365 ± 15 391 ± 18 394 ± 15 358 ± 28 — P =0.481 Triglyceride 84 ± 4 83 ± 4 94 ± 4 85 ± 3 — P = 0.207 Sinus Lesion369688 ± 32570 233056 ± 12746 245938 ± 20474 245750 ± 20423 225,714 ±5,869  P < 0.001 μm² Aorta lesion 4.5 5.4 4.5 8.3 1.4 P = 0.002 (% fromtotal area) Note: “Weight” is weight in grams; “Cholesterol” is plasmacholesterol and “Triglyceride” is plasma triglycerides, expressed inmg/dL.

As can be seen from FIG. 7, the results depicted in Table 5 demonstrateeffective inhibition of late stage atherogenesis measured in the tissuesof older mice following protracted oral exposure to a 1 mg/mouse dose ofthe D- and L-isomers of ALLE, and POVPC compared to PBS-fed controlmice. Oral administration of these compounds had no significant effecton other parameters measured, such as weight gain, total triglyceride orcholesterol plasma levels. Thus, the synthetic oxidized LDL componentsD-, L-ALLE and POVPC each individually exert anti-atherogenic activity,conferring nearly complete protection from atheromatous progression (ascompared with lesion scores at 24.5 weeks) in these geneticallysusceptible Apo-E KO mice. Surprisingly, it was observed that theinhibition of atherogenesis by these oxidized LDL analogs is accompaniedby a significant reduction in VLDL cholesterol and triglycerides, asmeasured by FPLC (FIGS. 8 and 9).

Example VII Inhibition of Atherogenesis in Genetically Predisposed(Apo-E KO) Mice by Oral Administration of CI-201

The ability of a stable form of an etherified phospholipid, the acidderivative of ALLE, CI-201, to suppress atherogenesis was investigated.Male 12 weeks old Apo-E KO mice were divided into two groups. In group A(n=14) mice were orally administered by gavage of CI-201 (0.025 mg/dose)suspended in PBS for 8 weeks every day (5 times a week). Mice in group B(n=15) received PBS (control). Atherosclerosis was assessed as describedabove. All mice were fed normal chow-diet containing 4.5% fat by weight(0.02% cholesterol) and water ad libitum.

As can be seen from FIG. 11, the results demonstrate a strikingattenuation of atherosclerotic progression measured in the tissues ofmice fed low doses of CI-201, as compared with unexposed control mice(PBS). Aortic sinus lesion in the CI-201 treated group was125,192±19,824 μm² and in the control group (PBS treated) was185,400±20,947 μm², demonstrating a decrease of 33% (P=0.051) of theaortic sinus lesion by oral administration of CI-201 in low dose. IL-10expression (determined by RT-PCR) in the aorta was higher by 40% in theCI-201 treated group, as compared with the control group. The elevatedexpression levels of IL-10 in the target organ, the aorta, support ananti-inflammatory effect induced by CI-201 oral administration. Thus,the stable synthetic oxidized LDL-201, exerts both oral-mediatedimmunomodulation and anti-inflammatory effect.

Example VIII Cytokine Expression in the Aorta of Apo-E KO Mice Treatedwith Oxidized Phospholipids (ALLE, CI-201, Et-Acetal, Me-Acetal & oxLDL)

The effect of ALLE, CI-201, its corresponding acetal derivativesEt-acetal and Me-acetal (Compounds IIa and IIb, FIG. 10) and oxLDL oncytokine expression in the target organ—the aorta—was evaluated usingRT-PCR as described hereinabove. Apo-E KO mice were orally administeredwith 1 mg/mouse ALLE, 1 mg/mouse CI-201, 1 mg/mouse Et-acetal, 1mg/mouse Me-acetal, 0.1 mg/mouse oxLDL or 0.2 ml/mouse PBS. Oraladministrations took place 5 times every other day. The expression ofthe anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokineIFN-γ and IL-12 were determined 8 weeks after final oral administration.

As can be seen in FIGS. 12 a and 12 b, mice treated with ALLE, CI-201,Et-acetal, Me-acetal and oxLDL showed elevated levels of IL-10expression as compared with the control PBS-treated group. As can beseen in FIGS. 12 c and 12 d, an opposite effect was shown in theexpression level of IFN-γ and IL-12. Reduced expression levels of IFN-γwas detectable in mice treated with ALLE, CI-201, Me-acetal and oxLDLand reduced levels of IL-12 was detectable in mice treated with ALLE,CI-201, Et-acetal and oxLDL.

Example IX Inhibition of Atherogenesis in LDL-RD Mice by Induction ofOral-Mediated Immunomodulation with oxLDL

In order to show that the synthetic oxidized phospholipids describedabove induce a similar effect as human oxidized LDL, a model forevaluating the effect of oxLDL on atherosclerosis progression in micewas designed.

LDL-RD Mice, 8-12 weeks old, were stratified by age, weight and lipidprofile (Cholesterol and Triglyceride) to different groups. Each groupwas treated with oxLDL in escalating doses (10, 100 or 1,000 μg/dosedissolved in PBS in a total volume of 0.2 ml PBS), albumin (100 μg/dosedissolved in PBS in a total volume of 0.2 ml PBS) or PBS (0.2 ml), 5times every other day. One day following the last oral administration,mice were challenged with an atherogenic diet (“Western diet”), adlibitum, and kept in 12 hours dark/light cycle, for five weeks.

Mice were sacrificed 6.5 weeks after the first oral administration andevaluated for the extent of atheromatous plaque area within the aorticsinus, as described hereinabove.

The effect of oxLDL treatment on metabolic parameters is delineated inTable 6 below. As is shown in Table 6, while oxLDL did not affect bodyweight or cholesterol levels, OxLDL in a dose of 100 μg/dosesignificantly (P<0.05) reduced the triglyceride levels as compared withthe PBS and albumin control groups.

TABLE 6 Effect of OxLDL treatment on Metabolic Parameters in LDL-RD miceGroup A Group B Group C Group D OxLDL OxLDL OxLDL Human Time 1,000 μg/100 μg/ 10 μg/ Serum Group E point Parameter dose dose dose albumin PBSStatistics T = 0 Weight (g) 28.1 ± 0.6 27.3 ± 0.6 26.9 ± 0.5 28.5 ± 0.627.9 ± 0.5 N.S* Cholesterol 161 ± 10 160 ± 8  156 ± 9  163 ± 10 162 ± 7 N.S* (mg/Dl) Triglyceride 154 ± 12 145 ± 11 152 ± 10 140 ± 14 155 ± 12N.S* (mg/Dl) END Weight(g) 29.0 ± 0.7 26.8 ± 0.4 27.6 ± 0.7 29.4 ± 1.829.1 ± 0.6 N.S* Cholesterol 1,541 ± 175  1,372 ± 122  1,458 ± 101  1,589± 76   1,554 ± 121  N.S* (mg/Dl) Triglyceride 344 120 250 303 306 P <0.001** (mg/Dl) *N.S: Not significant **Kruskal Wallis One Way Analysisof Variance on Ranks test was performed, data displayed as medianvalues. Note: “Weight” is weight in grams; “Cholesterol” is plasmacholesterol and “Triglyceride” is plasma triglycerides, expressed inmg/dL.

The attenuation of atherogenesis by oral administration of OxLDL isdemonstrated in FIG. 13 and in Table 7 below. As is shown in FIG. 13,treatment with both 100 μg/dose and 1,000 μg/dose oxLDL significantlydecreased (P<0.001) the lesion area in the aortic sinus by 45% ascompared with the control groups (PBS treated or human serum albumin(HAS) treated).

TABLE 7 effect of OxLDL treatment on atherosclerosis area in the aorticsinus Group A Group B Group C Group D OxLDL OxLDL OxLDL Human 1,000 μg/100 μg/ 10 μg/ Serum Group E Parameter dose dose dose albumin PBSStatistics Aortic Sinus 37,750 ± 4,890 38,304 ± 4,443 45,568 ± 3,30977,604 ± 5,039 69,712 ± 6,797 P < 0.001 Lesion (μm²)

Example X Inhibition of Atherogenesis in Genetically Predisposed (Apo-EKO) Mice by Oral Administration of CI-201

26-28 weeks old Apo-E KO mice (APO-E−/−<tm1Unc>[C57B/6J]) were used as aprevention of progression model. Mice were stratified by age, weight andlipid profile (cholesterol and triglyceride) to different groups. Onegroup was sacrificed at the beginning of the experiment and served asthe “base line” group. Each of the other groups was treated with CI-201in escalating doses (0.1, 1 or 10 μg/dose dissolved in PBS, 0.05%ethanol, in a total volume of 0.2 ml PBS). The control group receivedPBS (0.05% ethanol, 0.2 ml).

Mice were treated with CI-201 or PBS at three sets at the beginning ofeach month, each set consisted of 5 oral administrations every otherday. All mice were fed normal chow-diet containing 4.5% fat by weight(0.02% cholesterol) and water ad libitum and were kept in a 12 hoursdark/light cycle.

After 12 weeks mice were sacrificed and evaluated for the extent ofatheromatous plaque area within the aortic sinus, as describedhereinabove.

The effect of CI-201 treatment on metabolic parameters is presented inTable 8 below. The results show that CI-201 does not affect both thebody weight and the lipid profile of the tested mice.

TABLE 8 Effect of CI-201 treatment on Metabolic Parameters in Apo-E KOmice Group A Group B Group C Group E Time CI-201 CI-201 CI-201 Group DBase- point Parameter 10 μg/dose 1 μg/dose 0.1 μg/dose PBS LineStatistics T = 0 Weight (g) 26.6 ± 0.5 26.4 ± 0.4 26.4 ± 0.4 26.5 ± 0.426.7 ± 0.5 N.S* Cholesterol 321 ± 23 323 ± 20 313 ± 14 316 ± 12 315 ± 20N.S* (mg/Dl) Triglyceride 88 ± 4 87 ± 4 87 ± 6 80 ± 4 84 ± 8 N.S*(mg/Dl) END Weight(g) 29.6 ± 0.5 29.0 ± 0.4 28.8 ± 0.7 28.1 ± 1.8 — N.S*Cholesterol 344 ± 25 382 ± 24 406 ± 39 354 ± 24 — N.S* (mg/Dl)Triglyceride 76 ± 4 96 ± 8 107 ± 13 91 ± 7 — N.S* (mg/Dl) *N.S: Notsignificant Note: “Weight” is weight in grams; “Cholesterol” is plasmacholesterol and “Triglyceride” is plasma triglycerides, expressed inmg/dL.

The attenuation of atherogenesis by oral administration of CI-201 isdemonstrated in FIGS. 14 a-b and in Table 9 below.

TABLE 9 CI-201 effect on atherosclerosis area in the aortic sinus GroupA Group B Group C CI-201 CI-201 CI-201 Group E 10 μg/ 1 μg/ 0.1 μg/Group D Base- Parameter dose dose dose PBS Line Statistics Aortic Sinus272,483 ± 20,505 295,729 ± 20,909 228,000 ± 25,772 328,491 ± 21,920218,602 ± 29,248 P < 0.05 Lesion (μm²)

The results presented, in Table 9 show that CI-201 treatment completelyinhibited disease progression, such that the lesion areas in the aorticsinus of mice treated with different doses of CI-201 were similar tothat of the base line group.

Contrary to that, a 50% highly significant (p<0.01) increase inatherosclerotic lesion in the aortic sinus was observed in the PBStreated mice as compared with the base line group (328,491±21,920 μm² inthe PBS group versus 218,602±29,248 μm² in the base line group).

As is demonstrated in FIG. 14 a, all doses of CI-201 inhibited thedisease progression, while the most effective dose was the minimal doseof 0.1 μg/dose. As is demonstrated in FIG. 14 b, the group treated with0.1 μg/dose CI-201 exhibit a 92% significant (P<0.05) decrease inatherosclerotic lesion in the aortic sinus as compared with the PBStreated group (328,491±21,920 μm² in the PBS group versus 228,000±25,772μm² in the CI-201 treated group).

Example XI Elevation of Inflammation Markers in the Serum of Apo-E KOMice Treated with CI-201

In view of the dramatic inhibition of atherosclerosis progression byoral administration of CI-201, which, as described hereinabove, is notattributed to alteration of body weight or lipid profile inducedthereby, the effect of oral administration of CI-201 on the level ofinflammation markers in the serum was evaluated, in order to investigateits mechanism of action.

As is shown in experimental and clinical studies, IL-10 is a majorprotective cytokine in plaque growth and stability. For example,Caligiuri et al. (Interleukin-10 deficiency increases atherosclerosis,thrombosis, and low-density lipoproteins in apolipoprotein E knockoutmice. Mol. Med. 2003; 9(1-2):10-17) recently reported that lesion sizewas dramatically increased in double KO mice for Apo-E and IL-10, ascompared with controls, and the proteolitic and procoagulant activitywere elevated, showing that IL-10 may reduce atherosclerosis and improvethe stability of plaques.

Another marker of acute inflammatory state is Serum Amyloide A (SAA), ahigh sensitive inflammatory marker which can be increased up to 1,000fold during inflammation. SAA as a CRP (C Reactive Protein) issynthesized by the liver in response to IL-1, IL-6 and TNF (Balke andRidker, Novel clinical markers of vascular wall inflammation, Circ Res.2001; 89:763-771.). SAA has been found to be is expressed by severalcell types in atherosclerotic lesion (Meek et al. Expression ofapolipoprotein serum amyloid A mRNA in human atherosclerotic lesions andcultured vascular cells: implications for serum amyloid A function. ProcNatl Acad Sci USA 1994; 91:3186-3190; Uhlar and Whitehead. Serum amyloidA, the major vertebrate acute-phase reactant. Eur J Biochem. 1999;265:501-523).

Initiation of the inflammatory cascade occurs primarily throughactivated blood monocytes and tissue macrophages at the site of theinflammatory stimulus. Upon activation macrophages release a range ofprimary inflammatory mediators, the most important of which are membersof the IL-1 and TNF cytokine families, which trigger the release of arange of secondary cytokines and chemokines (IL-6, IL-8 and MCP). Thechemotactic activities of these molecules draw leukocytes to theinflammatory site, where they in turn release further pro-inflammatorycytokines.

Thus, Apo-E KO mice were orally administered with 0.1 μg/mouse CI-201 or0.2 ml/mouse PBS, 5 times every other day. Mice serum was collectedbefore treatment (day 0), at the end of the treatment (two weeks) andtwo weeks thereafter (4 weeks) and the level of the inflammation markersIL-10 and Serum Amyloide A (SAA) were evaluated.

The obtained data are presented in FIG. 15 a (for IL-10 levels) and FIG.15 b (for SAA levels).

As can be seen in FIG. 15 a, at the end of the treatment (2 weeks), asubstantial increase in IL-10 serum level was observed, while 2 weeksthereafter (4 weeks) a decay has been noticed. In the control,PBS-treated group, no change in IL-10 serum levels was noticedthroughout the experiment.

As can be seen in FIG. 15 b, while SAA serum levels dramaticallyincreased in the control group, no alterations in SAA serum levels inthe CI-201 treated group were observed.

These results clearly indicate that by elevating IL-10 levels in theserum, CI-201 induce an anti-inflammatory response that may shut down apro-inflammatory response, demonstrated by elevated levels of SAA.Systemic inflammation manifested by high SAA may promote atheroscleroticplaque destabilization in addition to exerting a possible direct effecton atherogenesis. These results further suggest a direct effect ofCI-201 on inflammatory processes.

Example XII Cytokine Expression in Various Organs of Apo-E KO MiceTreated with CI-201

The effects of CI-201 treatment on cytokine expression in the targetedorgan—the aorta, as well as in the spleen, liver, kidneys and smallintestine were evaluated using RT-PCR as described hereinabove. Apo-E KOmice were orally administered with 1 mg/dose CI-201 or with 0.2 ml/mousePBS, 5 times every other day. The expression of the anti-inflammatorycytokine IL-10 and the pro-inflammatory cytokine IFN-γ were determined 8weeks after final oral administration. The data obtained are presentedin FIGS. 16 a-b and FIG. 17.

As can be seen in FIGS. 16 a and 16 b, mice treated with CI-201 showedelevated levels of the anti-inflammatory cytokine IL-10 as compared withthe control PBS-treated group, while an opposite effect, namely, reducedexpression level, was shown in the expression level of thepro-inflammatory cytokine IFN-γ in the CI-201 treated group.

The increase in the anti-inflammatory response, as demonstrated byelevated levels of IL-10, accompanied with decreased pro-inflammatoryresponse, as demonstrated by decreased levels of IFN-γ, furtheremphasize the immunomodulation induced by CI-201, which is effected byswitching from Th1 towards Th2 response within the aorta, as well as theanti-inflammatory effect thereof.

While in the targeted organ, the aorta, CI-201 increases theanti-inflammatory response, such an effect was not observed in otherorgans. As can be seen in FIG. 17, no differences were observed incytokine expression in the spleen and in the small intestine between theCI-201 treated group and the control, PBS treated, group. It issuggested that the Peyers patches located therein encountered theorally-administered antigen. No change in cytokine expression wasobserved in the liver and in kidney as well (data not shown).

The results above suggest that the oxidized phospholipids analogs of thepresent invention inhibit atherosclerosis via a pathway that affectsboth the immune system and inflammation. However, it is possible thatother mechanisms are also involved in the most potent inhibitory effectthereof.

Example XIII Inhibition of Rheumatoid Arthritis in AdjuvantArthritis-Induced Rats by Oral Administration of CI-201

Rheumatoid arthritis (RA) is a severe autoimmune disease involvingchronic joint inflammation and destruction. Adjuvant-induced arthritis(AIA) is the first experimental arthritis model (Pearson. Development ofarthritis, periarthritis and periostitis in rats given adjuvant. ProcSoc Exp Biol Med 1956; 91:95-101; Pearson and Wood. Studies ofpolyarthritis and other lesions induced in rats by injection ofmycobacterial adjuvant. I. general clinical and pathologiccharacteristics and some modifying factors. Arthritis Rheum 1959;2:440-459).

The morphologic character of early AIA lesions is based on cell-mediatedimmunity (CMI). Lymphocytes infiltration is followed by edema, fibrindeposition, and necrosis, accompanied by proliferation of synoviocytesand fibroblasts and activation of osteoblasts and osteoclasts. Theinflammatory infiltrate in the joint lesions of AIA contain T cellsactivated by specific antigens. Th1 cytokines, such as IL-17, IFN-γ, andTNF-α, are expressed in early AIA together with cytokinescharacteristics of macrophage activation. In a later phase of thedisease, levels of IL-4, IL-6 and JE (murine homologe of monocytechemoattractant protein 1) and TGF-β are elevated. There is localrelease of proteolytic enzymes and/or free radicals of oxygen, whichresults in a progressive breakdown of collagen type II and IX, matrixdamage, and in time, degradation of bone (Van Eden and Waksman. Immuneregulation in adjuvant-induced arthritis. Possible implications forinnovative therapeutic strategies in arthritis. Arthritis Rheum 2003;48(7):1788-1796).

Several attempts at immunotherapy of human autoimmune diseases such asrheumatoid arthritis (RA), type I diabetes, and multiple sclerosis,based either on modulation of individual immune pathways involved ininflammation or on tolerization to various antigens, have shown thatthis approach may be viable (Bielekova et al. Encephalitogenic potentialof the myelin basic protein peptide (amino acids 83-99) in multiplesclerosis: Results of a phase II clinical trial with an altered peptideligand. Nat Med. 2000; 6:1167-1175; Kappos et al. Induction of anon-encephalitogenic type 2 T helper-cell autoimmune response inmultiple sclerosis after administration of an altered peptide ligand ina placebo-controlled, randomized phase II trial. Nat. Med. 2000;6:1176-1182).

In many patients, the outcome of Rheumatoid Arthritis is complicated bycardiovascular disease, the latter being the main cause of the increasedmortality in this disorder. Autoantibodies against cardiolipin (CL) andoxidatively modified low density lipoproteins (copper oxidized LDL),including malondialdehyde modified LDL (MDA-LDL), have been suggested tohave a predictive value for cardiovascular disease. It has beendemonstrated that there are increased levels of autoantibodies againstcopper-oxidized low density lipoprotein, malondialdehyde-modified lowdensity lipoproteins and cardiolipin in patients with rheumatoidarthritis (Cvetkovic et al. Increased levels of autoantibodies againstcopper-oxidized low density lipoprotein, malondialdehyde-modified lowdensity lipoprotein and cardiolipin in patients with rheumatoidarthritis. Rheumatology. 2002; 41:988-995). Moreover there are evidencesfor oxidized low density lipoprotein in synovial fluid from rheumatoidarthritis patients (Dai et al. Evidence for oxidized low densitylipoprotein in synovial fluid from rheumatoid arthritis patients. FreeRadic Res. 2000; 32(6):479-486).

Since CI-201 was found to be effective in both induction ofimmunomodulation to Ox LDL and increasing an anti-inflammatory response,its effect on arthritis development was tested.

Nine weeks old male Lewis rats were orally administered with differentdoses of CI-201 (4 mg/kg or 0.4 mg/kg) or with PBS, 5 times every otherday. Adjuvant arthritis was then induced by intradermal injection of 0.1ml of tuberculosis suspension. Intensity of arthritis was monitored bymeasuring paw swelling while mobility of the animals was monitored aswell. The study design is presented in FIG. 18. The results arepresented in FIG. 19.

As can be seen in FIG. 19, pre-treatment with the higher dose (4.0mg/kg) of CI-201 resulted in a significant decrease in rats pawswelling, as compared with the control, PBS-pretreated rats.

While the PBS treated rats were barely moving, using only their backlegs, the mobility of rats pre-treated with the higher dose of CI-201was close to that of normal rats.

In order to evaluate the effect of a continuous treatment with CI-201 onAIA-induced Lewis rats, following the AIA-induction, 9 weeks old maleLewis rats were fed, 5 times every other day, before induction of AIA byintradermal injection of 0.1 ml of tuberculosis suspension and werethereafter continuously fed, three times a week, for about 30 days. Thestudy design is presented in FIG. 20. The results are presented in FIG.21-23.

As can be seen in FIGS. 21-23 CI-201, a continuous treatment with highdose of CI-201 substantially attenuated arthritis development in all thetested parameters.

These results clearly indicate that the anti-inflammatory properties ofCI-201 can further influence a classic inflammatory disease, RA, inaddition to its effect on atherosclerosis.

CD4+ T-helper cells and macrophages infiltrate the synovial membrane(SM) in chronic, destructive rheumatoid arthritis and probably play acentral role in promoting and maintaining the disease process. CD4+ Tcells can differentiate into Th1 subpopulation, characterized bypredominant production of IFN-γ. Predominance of pro-inflammatory Th1type cells has been postulated in RA (Schmidt-Weber et al. Cytokine geneactivation in synovial membrane, regional lymph nodes, and spleen duringthe course of rat adjuvant arthritis. Cell. Immunol. 1999; 195:53-65.).Macrophages are also highly activated in the inflammatory process in RA,both locally and systemically.

The resemblances in the inflammatory response involved both inatherosclerosis and arthritis support the suggestion that CI-201 inducesan anti-inflammatory response in AIA similarly to that demonstratedabove in atherosclerosis.

It can therefore be postulated that CI-201 treatment induce IL-10elevation in AIA model and IL-10 can suppress pro-inflammatory cytokine,thus reducing the disease outcome, as was demonstrated by decreased pawswelling and better mobility. These results therefore implicate that theoxidized phospholipids analogs of the present invention can serve as anew family of therapeutic drug for treating Rheumatoid Arthritis, aswell as other autoimmune and/or inflammatory diseases.

Example XIV Oral Administration of the Pre-Oxidized Compound V toGenetically Predisposed (Apo-E KO) Mice The Effect of an Oxidized Groupon Atherogenesis Inhibition

The effect of the oxidized group in ALLE and CI-201 was tested bycomparing the effect of oral administration of ALLE and CI-201 on earlyatherogenesis and progression of advanced atherosclerotic plaques, shownabove, with the effect of the pre-oxidized derivative thereof. CompoundV (1-Hexadecyl-2-(5′-hexenyl)-sn-glycero-3-phosphocholine, Example I).

25 female, 8-10 week old Apo-E KO mice were divided into 4 groups. Eachgroup was fed with 5 mg/mouse Compound V suspended in 0.2 ml PBS (GroupA, n=6), 1 mg/mouse Compound V″ suspended in 0.2 ml PBS (Group B, n=6),0.2 mg/mouse Compound V suspended in 0.2 ml PBS (Group C, n=6), and PBS(Group D, control, n=7), every other day for 5 days. Eight weeks afterthe last oral administration mice were sacrificed. Mice were bled priorto feeding (Time 0) and at the conclusion of the experiment (End) fordetermination of lipid profile. Atherosclerosis was assessed in theheart, as described hereinabove. All mice were fed normal chow-dietcontaining 4.5% fat by weight (0.02% cholesterol) and water ad libitum.

The effect of Compound V treatment on metabolic parameters and onatherogenesis is delineated in Table 10 below. The effect of Compound Von atherogenesis is further presented in FIG. 24.

TABLE 10 The effect of the pre-oxidized derivative Compound V onmetabolic parameters and atherosclerosis area in the aortic sinus inApoE KO mice 5 mg 1 mg 0.2 mg Time Parameter PBS Comp. V Comp. V Comp. VStatistics point tested (n = 7) (n = 6) (n = 6) (n = 6) (p value) 0Weight 18.4 ± 0.4 18.0 ± 0.3 18.3 ± 0.3 18.5 ± 0.3 0.826 Cholesterol 197± 19 210 ± 18 198 ± 23 214 ± 21 0.902 Triglyceride  53 ± 10 52 ± 4 52 ±5 50 ± 7 0.993 End Weight 20.7 ± 0.4 19.8 ± 0.3 19.8 ± 0.2 20.1 ± 0.30.161 Cholesterol 387 ± 20 431 ± 23 398 ± 23 409 ± 8  0.455 Triglyceride70 ± 3 80 ± 8  91 ± 4** 102 ± 5* 0.001 Sinus Lesion 314764 ± 14458307291 ± 22689 361166 ± 24068 334622 ± 26100 0.352 μm² Note: “Weight” isweight in grams; “Cholesterol” is serum cholesterol and “Triglyceride”is serum triglycerides, expressed in mg/dL. *P < 0.001 versus the PBSgroup. **P < 0.05 versus the PBS group.

As can be clearly seen in FIG. 24, while oral administration of theoxidized compounds CI-201 and ALLE substantially inhibited atherogenesisin Apo-E KO mice, no effect on atherogenesis was observed followingtreatment with the pre-oxidized derivative Compound V, thus indicatingthe importance of the presence of the oxidized group in treatingatherogenesis.

Example XV Oral Administration of the Pre-Oxidized Compound V toGenetically Predisposed (Apo-E KO) Mice The Effect of an Oxidized Groupon Atherogenesis Progression

The pre-oxidized Compound V was further tested in the prevention ofprogression model in ApoE KO mice, described hereinabove. 23-26 weeksold Apo-E KO mice (APO-E−/−<tm1Unc>[C57B/6J]) were stratified by age,weight and lipid profile (cholesterol and triglyceride) to differentgroups. One group was sacrificed at the beginning of the experiment andserved as the “base line” group (B.L., n=10). The second group wastreated with Compound V (0.1 μg/dose, n=10) dissolved in PBS, 0.05%ethanol, in a total volume of 0.2 ml PBS. The control group received PBS(0.05% ethanol, 0.2 ml) (n=11).

Mice were treated with compound V or PBS at three sets at the beginningof each month, each set consisted of 5 oral administrations every otherday. All mice were fed normal chow-diet containing 4.5% fat by weight(0.02% cholesterol) and water ad libitum and were kept in a 12 hoursdark/light cycle.

After 12 weeks, mice were sacrificed and evaluated for lipid profile andextent of atheromatous plaque area within the aortic sinus, as describedherein above. The results are presented in Table 11 below and in FIG. 25and clearly indicate that oral administration of Compound V did noteffect atherogenesis progression.

TABLE 11 The effect of the pre-oxidized derivative Compound V onmetabolic parameters and atherosclerosis area in the aortic sinus inApoE KO mice Time Comp. V Statistics point Parameter 0.1 μg/dose PBSBase-Line (p value) T = 0 Weight (g) 26.1 ± 0.7 26.2 ± 0.6 26.2 ± 0.70.710 Cholesterol 401 ± 27 393 ± 32 406 ± 19 0.936 (mg/Dl) Triglyceride128 ± 8  129 ± 6  125 ± 7  0.926 (mg/Dl) END Weight (g) 28.1 ± 0.5 28.0± 0.5 — 0.967 Cholesterol 292 ± 16 277 ± 37 — 0.717 (mg/Dl) Triglyceride81 ± 3 90 ± 4 — 0.094 (mg/Dl) Sinus Lesion μm² 238156 ± 32206 206647 ±15293 137451 ± 18975 0.011* *There is no statistical difference betweenthe compound V treated mice and the control mice, PBS treated.

Example XVI The In Vitro Effect of CI-201 on Dendritic Cells

Materials and Methods

Mice: Female 8-10 week old C67BL/6 and SJL mice were purchased from theHarlan laboratories, Israel.

Phospholipids: The molecules tested in vitro for inhibition of DCcytokine secretion were:1-O-hexadecyl-2-(4′-carboxybutyl)-sn-glycero-3-phosphocholine[(R)—CI-201], (S)—CI-201, Racemic CI-201, Methyl ester of CI-201,Non-phosphorylated metabolite (NPM) of CI-201,1-O-Hexadecyl-2-(5′-Hexenyl)-sn-glycero-3-phosphocholine (Compound V)and 1-O-Hexadecyl-2-(4′-carboxybutyl)-sn-Glycero-3-Phosphoethanolamineall were synthesized in VBL's chemical laboratory, Or Yehuda, Israel).1-O-Hexadecyl-2-Acetoyl-sn-Glycero-3-Phosphocholine (PAF),L-a-Phosphatidylcholine, and Valeric acid were from Sigma-Aldrich(Rehovot, Israel).1-Palmitoyl-2-(5′-oxo-Valeroyl)-sn-Glycero-3-Phosphocholine (POVPC),1-Palmitoyl-2-Glutaroyl-sn-Glycero-3-Phosphocholine (PGPC),1-O-Octadecyl-2-Acetoyl-sn-Glycero-3-Phosphocholine (C18 PAF),1-O-Hexadecyl-2-O-Butyroyl-sn-Glycero-3-Phosphocholine (PAF-2C),1-O-Palmitoyl-2-Acetoyl-sn-Glycero-3-Phosphocholine (PAF ester) and1-O-Hexadecyl-2-Azelaoyl-sn-Glycero-3-Phosphocholine (azPC) werepurchased from Avanti Polar Lipids (Alabaster, Ala.).

Isolation of bone-marrow derived dendritic cells (BMDC's): Bone-marrowwas flushed out with cold RPMI-1640 from mice femur and tibia. A cellsuspension was prepared and erythrocytes were removed using red bloodcell (RBC) lysis buffer (Beit Haemek, Israel). Cells were washed inphosphate buffer saline (PBS) (Beit Haemek, Israel), and incubated at 4°C. for 15 minutes in buffer containing PBS and 0.5% bovine serum albumin(BSA) with mouse B220 and CD90 microbeads (Miltenyi biotech, BergischGladbach, Germany). Cells were then washed, resuspended in the samebuffer, and depleted from B and T cells on a Midi-Macs separation unitthrough a LD or LS column (Miltenyi biotech). The depleted cells werethen counted, washed and seeded (10⁶/ml) in medium containing RPMI-1640,L-glutamine, β-mercaptoethanol, 10% FCS, antibiotics (Penicillin,Streptomycin) and 20 ng/ml of mouse GM-CSF (Peprotech, Rehovot, Israel).Medium was replaced every other day and cells were used for subsequentexperiments on days 5-6 post culturing.

Isolation of Monocytes/Macrophages: Spleens were removed and a cellsuspension was prepared. Erythrocytes were depleted using red blood cell(RBC) lysis buffer (Beit Haemek, Israel). Cells were washed in phosphatebuffer saline (PBS) (Beit Haemek, Israel), and incubated in 4° C. for 15min in buffer containing PBS and 0.5% bovine serum albumin (BSA) withmouse CD11b microbeads (Miltenyi biotech, Bergisch Gladbach, Germany).Cells were then washed, resuspended in the same buffer and positivelyselected on a Mini-Macs separation unit through a MS column (Miltenyibiotech).

Isolation of human monocyte derived dendritic cells (Mo-DC's): PBMCswere isolated from heparinized whole blood from healthy donors byFicoll-Paque (Amersham, Uppsala, Sweden). Cells were incubated at 4° C.for 15 minutes in buffer containing PBS, 0.5% bovine serum albumin (BSA)and 2 mM EDTA with human CD14 microbeads (Miltenyi biotech) washed andpositively selected on a Midi-Macs separation unit through LS column.The resulting cells were counted washed and seeded (10⁶/ml) in mediumcontaining RPMI-1640, L-glutamine, β-mercaptoethanol, 10% FCS,antibiotics (Penicillin, Streptomycin), non essential amino acids,sodium pyruvate, 40 ng/ml of human GM-CSF and 20 ng/ml of human IL-4(R&D systems, Minneapolis, Minn.). Medium was replaced every other dayand cells were used for subsequent experiments on days 5-6 postculturing. Cell purity was >75% as determined by FACS for CD11c.

Cytokine analysis: For the detection of cytokine production,supernatants from BMDC's and Mo-DC's following a 24 hour treatment weretested with Duoset ELISA kits for mouse IL12p40, TNF-α, IL-6 and humanIL12p40 and TNF-α (R&D systems, Minneapolis, Minn.) respectively.

For measurement of IFN-γ by ex-vivo activated T cells, SJL female micewere orally administered with CI-201 (10 and 100 μg/mouse/feed) or PBSas control, once a day, every other day for a total of 5 doses (dosingvolume was 0.2 ml) and then immunized subcutaneously in two flanks with200 μl of emulsion containing 200 μg of PLP peptide 139-151 and 400 μgof MT followed by 4 more administrations every other day. Lymph nodeswere collected 10 days later, stimulated ex vivo with 10 μg/ml of PLP139-151 peptide or 0.5 μg/ml of anti CD3 and three days later cytokineproduction from the supernatant was measured using the Duoset ELISA kit(R&D systems, Minneapolis, Minn.).

Ex-vivo ELISA and Intracellular staining: For intracellular staining,C57BL/6 were orally administered with CI-201 (0.1 μg/mouse/feed) or PBSas control, 5 times, once every other day. Dosing volume was 0.2 ml.Subsequently, mice were immunized subcutaneously in two flanks with 200μl of emulsion containing 200 μg of ovalbumin and MT (100 μg). Ten dayslater, during which alternate oral feedings of PBS and CI-201 werecontinued, mice were sacrificed, a cell suspension was prepared from thedraining lymph nodes and cells were activated ex vivo with 1 mg/ml ofovalbumin. One week later, cells were re-stimulated with plate-boundanti CD3 and anti CD28 and tested for cytokine production byintracellular staining. Results are on gated CD4+ T cells. Allantibodies used (IL-4-APC, IFN-γ-PE, TNF-α-APC, IL-10-PE, CD4-FITC) arefrom Pharmingen (San Diego, Calif.).

Preparation of RNA, cDNA and Quantitative Real Time PCR (Q-PCR): RNA wasprepared from cells using RNeasy mini kit (Qiagen, Valencia, Calif.).For cDNA preparation, 1 μg of RNA was combined with Oligo dT for 10minutes at 70° C., 1^(st) strand buffer, DTT and dNTP and super-scriptreverse transcriptase (SS-II) (Invitrogen, Carlsbad, Calif.) were addedfor 50 minutes at 42° C. and the reaction was ended by incubation for anadditional 15 minutes at 70° C. All real time PCR reactions wereperformed using lightcycler Taqman master (Roche diagnostics, Mannheim,Germany) and run on the LightCycler machine (Roche). Q-PCR was done witha set of matched primers (Sigma-Genosys, Israel) and probes (Rochediagnostics, Mannheim, Germany) as was suggested in Roche's web site:www.roche-applied-science.com/servlet for mouse IL27p28(5′-CATGGCATCACCTCTCTGAC, SEQ ID NO: 1), (3′-AAGGGCCGAAGTGTGGTAG, SEQ IDNO: 2), probe #38 Cat#04687965001, IL12p35 (5′-GAGACTTCTTCCACAACAAGAGG,SEQ ID NO: 3), (3′-CTACCAAGGCACAGGGTCAT, SEQ ID NO: 4) probe #27cat#04687582001 and IL23p19 (5′-CACCAGCGGGACATATGAA, SEQ ID NO: 5),(3′-CCTTGTGGGTCACAACCAT, SEQ ID NO: 6), prone #47 cat#04688074001. Readysets of probe with primer were used for IL12p40 and GAPDH assays(Applied Biosystems, assays #Mm01288992_ml and Mm99999915_g1respectively) with the latter served to normalize RNA levels.

Cell stimulation: Mouse BMDC's were activated with 100 ng/ml E. coli LPS05:55 (Sigma-Aldrich), 10 μg/ml peptidoglycan (PGN), 25 μg/ml Poly I:C,300 ng/ml Pam₃CSK4 and 1 μg/ml Fc-soluble CD40L (InvivoGen, San Diego,Calif.). Cells were pre-incubated for 1 hour with various phospholipidsat the indicated concentrations before activation with TLR ligands orFc-soluble CD40L and supernatants collected 24 hours later. HumanMo-DC's were activated with PGN. For PBMC's and CD14+, cells were preincubated with human IFN-γ (20 ng/ml) and maintained during a 24 houractivation. For RNA preparation, cells were pre incubated for 1 hourwith 20 μg/ml of CI-201 and then activated for the indicated time pointswith PGN before collection. In the experiments conducted to determinepotential active sites, phospholipids were added at the indicatedconcentrations 1 hour prior to activation with PGN.

Gene Chip analysis: Mouse BMDC's were enriched for CD11c+ DC (>90%) withmouse CD11c microbeads from 5-6 days cultures over MS or LS columns(Miltenyi biotech). CD11c+ DC were stimulated for 3 hours with 10 μg/mlPGN alone or in the presence of 20 μg/ml of CI-201. RNA was prepared andused to generate cRNA. Preparation of cRNA, hybridization and scanningwas done in the Weizmann Institute of Science (Rehovot, Israel) on themouse genome 430A 2.0 array (Affymetrix, Santa Clara, Calif.).

XV1A: CI-201 Inhibits IL12p40 but not TNF-α, IL-6 and IL-1 Productionfrom Activated BMDC's

As indicated above, oral administration of CI-201 reduces the level ofIFN-γ messenger RNA in the aortas of ApoE−/− mice. As T cells are amajor source of IFN-γ and Th1 cells were shown to play a role inatherosclerosis, the effect of CI-201 treatment on IFN-γ production byCD4+ T cells was assessed.

Results

The data presented in FIGS. 26A-B demonstrates that in vivo treatmentwith CI-201 resulted in reduced IFN-γ production by CD4+ T cells.

In light of the above, it was speculated that CI-201 might have a directinhibitory effect on IFN-γ production by T cells. To address that,radiolabeled CI-201 was incubated with different immune cell lines andthe level of incorporation was measured. Unexpectedly, the highestCI-201 incorporation was detected in DC's and macrophages, and not in Tcells, which showed a mild increase in radioactive levels only at the 24hours time point compared to control cells (FIG. 27). Accordingly, itwas hypothesized that CI-201 impairs IFN-γ production by CD4+ T cellsthrough regulation of IL12p40 (p40) secretion by APC's.

To test that, BMDC's were activated with several TLR antagonists in thepresence of varying concentrations of CI-201. Supernatants collected 24hours later were assayed for the production of p40 by ELISA. FIGS. 28A-Fdemonstrate that IL12p40 production was inhibited to various degrees byCI-201 treatment following activation of BMDC's with the different TLRligands used. When BMDC'S were activated with PGN (TLR 2/6), asignificant and dose dependent reduction in p40 production was observed(FIG. 28A), while activation with another TLR 2 ligand, Pam3CSK4(TLR1/2) resulted in significant p40 inhibition (FIG. 28B).

The effect on p40 production was then tested following activation withLPS, an agonist to TLR4 that signals downstream through both Myd88dependent and independent pathways. A significant, but small reductionof p40 levels were detected in the presence of intermediatedconcentrations of CI-201 (FIG. 28C).

Next, the effect of CI-201 on p40 production from DC's activated throughintracellular TLR was investigated. While a significant and dosedependent p40 inhibition with CI-201 could be noticed following PolyI:C(TLR3) activation (Myd88 independent) (FIG. 28D), only 20 μg/ml ofCI-201 was able to significantly inhibit p40 secretion after CpG (TLR9)engagement (FIG. 28E). To determine whether CI-201 conveys itsinhibitory function via interfering with TLR binding, BMDC's wereactivated with Fc-CD40L. This induces cytokine production by BMDC's in aTLR, MyD88-independent pathway. As shown in FIG. 28F, Fc-CD40Lactivation in the presence of CI-201 resulted in a significant reductionof p40 secretion by BMDC's.

Because activation of BMDC's through TLR's and CD40 is known to inducethe production of cytokines other than p40, TNF-α levels were measuredfollowing activation with all TLR agonists used above. In addition, IL-6and IL-1 levels were also measured following PGN DC's activation in thepresence of CI-201. The results presented in FIGS. 28G-M demonstratethat except for PolyI:C, CI-201 did not impair the production of TNF-αby other activators, and likewise did not impair the production of IL-6and IL-1 following PGN activation of BMDC's. Taken together, theseresults suggest that CI-201 impedes a particular pathway responsible forthe induction of p40 production while leaving pathways regulating acuteproinflammatory cytokine production unaffected.

XV1B: Gene Chip and Real-Time PCR Analysis

In order to determine p40's level of regulation and to reveal putativeadditional modified genes subsequent to CI-201 treatment, gene chipanalysis was conducted on purified CD11c+ BMDC's activated for 3 hourswith PGN in the presence of CI-201.

Results

Several genes were found to be up-regulated and 5 genes, including p40had a reduced RNA expression. In order to confirm the gene chip resultsand examine p40's regulation kinetics by CI-201, quantitative real timePCR was performed for p40 and for the paired chains constitutingIL12(p35), IL-23(p19) and IL-27(p28) with the latter recently shown toplay a role in IFN-γ production by T cells as well. Similar to theresults obtained from the gene chip analysis, CI-201 clearly inhibitedp40 RNA expression in the first 6 hours following activation (FIG. 29A)by more than two folds compared to control. It was also discovered thattreatment with CI-201 resulted in a substantial inhibition of the RNAexpression level of p19 (FIG. 29C). Following reevaluation of the genechip results, a reduced p19 expression in the CI-201 treated cells wasindeed observed, thereby confirming the real time PCR results. CI-201did not modify the RNA expression levels of p35 and p28 (FIGS. 29B and29D). These results indicate that CI-201 regulates the RNA expressionlevels of IL-12 and IL-23, two key pro-inflammatory cytokines shown toplay a pivotal role in inflammatory diseases.

XV1C: CI-201 Alters p40 Production by Mo-DC

Since CI-201 leads to the reduction of p40 expression from activatedBMDC's the effect of CI-201 on human cells was also examined. Mo-DC'swere activated with PGN in the presence of CI-201 and the supernatantswere tested for p40 and TNF-α production.

Results

The results depicted in FIG. 30A indicate that CI-201 can inhibit p40production by activated Mo-DC's without altering the cells ability toproduce TNF-α (FIG. 30B).

XV1C: CI-201 Inhibits p40 Production by PBMC but not byMonocytes/Macrophages

The data presented above clearly demonstrate that CI-201 can regulatep40 production by DC's. The next experiment was performed in order totest whether p40 inhibition could be detected when CI-201 is applied ontotal PBMC's and monocytes/macrophages.

Results

PBMC's from healthy donors were incubated over-night with IFN-γ and thenactivated with PGN in the presence of CI-201 for another 24 hours.Supernatants were then evaluated for p40 and TNF-α production. Asobserved with DC's, CI-201 inhibited p40 production when applied to PGNactivated PBMC's from two different donors. However, unlike with DC's, aclose to complete inhibition of p40 production could be achieved in allCI-201's tested concentrations (FIGS. 31A-B). In agreement with the DCdata above, CI-201 did not impair TNF-α production by PBMC's from donorI while a modest reduction in TNF-α levels was measured from donor IIPBMC's (FIGS. 31C-D).

In order to assess CI-201 effect on p40 production by macrophages,splenic CD 11b cells were incubated over night with IFN-γ and thenactivated for 24 hours with PGN in the presence CI-201. Whensupernatants were evaluated for p40, no major differences in p40 andTNF-α production levels were observed between control and CI-201 treatedcells suggesting that CI-201 may selectively act on DC's (FIGS. 32A-B).

XV1D: Structure-Function Studies with CI-201

The in vitro experiments described hereinabove demonstrated that p40production by activated DC's could be maximally inhibited by the highestconcentration of CI-201 used (20 μg/ml). However, based on previousexperience with small molecules designed for therapeutic applications,it is highly desirable to reach beneficial effect with lower substanceconcentration in the range of ng/ml. Therefore, it was a priority todesign a superior molecule on the backbone of CI-201 that will exhibitsimilar in vitro effect on p40 production when applied to DC's. Toattain such a molecule, it is important to establish the residue/s whichare essential for CI-201's functionality.

Results

In a set of experiments, BMDC's were activated with PGN in the presenceof commercially available and self synthesized oxidized and non oxidizedphospholipids, with p40 production levels placed as the measuredparameter to determine inhibitory efficacy. Preliminary resultsindicated that several facets such as the length, the chemicalcomposition and the chemical bonds of each chain should be considered infuture molecular designs. Thus, replacement of the hydrogen group for amethyl group in R2 (methyl ester of CI-201) and removal of thephosphocholine group in R3 (non phosphorylated metabolite of CI-201)restored p40 production (FIGS. 33B-C). Nonetheless, exchange of thethree methyl groups in R3 for three hydrogen molecules (phosphoethanolamine) did not alter the molecule's p40 inhibitory effect (FIG. 33D). Agroup of molecules, some closely resembling the structure of CI-201(POVPC and PVPC), were tested, all bearing an esteric bond on R1 and R2instead of the etheric bonds found in CI-201. A third group of moleculeswas also selected for testing that were derived from PAF, which has somesimilarities to CI-201. It was found that PAF itself and PAF2C with anextended R2 molecule were efficient in p40 inhibition (FIGS. 33H-I).Shortening of R2 mildly reduced the activity of PAF (FIG. 33K), but anadditional esteric bond in R1, elongation by 2 methyl group of R2 or along R2 with oxidized edge, all resulted in loss of p40 inhibition(FIGS. 33L-M).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

ADDITIONAL REFERENCES OF INTEREST Not Cited within the Text

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1. A method of treating or preventing an inflammation associated with anendogenous oxidized lipid, the method comprising administering to asubject in need thereof a therapeutically effective amount of at leastone oxidized lipid, thereby treating or preventing the inflammatorydisease or disorder associated with an endogenous oxidized lipid in saidsubject.
 2. The method of claim 1, wherein said oxidized lipid isselected from the group consisting of an oxidized phospholipid, aplatelet activating factor, a plasmalogen, a substituted orunsubstituted 3-30 carbon atoms hydrocarbon terminating with an oxidizedgroup, an oxidized sphingolipid, an oxidized glycolipid, an oxidizedmembrane lipid, and any analog or derivative thereof.
 3. The method ofclaim 1, wherein said oxidized lipid has the general formula I:

wherein: n is an integer of 1-6, whereas if n=1, Cn, Bn, Rn and Y areabsent; each of B₁, B₂, . . . Bn−1 and Bn is independently selected fromthe group consisting of oxygen, sulfur, nitrogen, phosphor and silicon,whereby each of said nitrogen, phosphor and silicon is substituted by atleast one substituent selected from the group consisting of hydrogen,lone pair electrons, alkyl, halo, cycloalkyl, aryl, hydroxy,thiohydroxy, alkoxy, aryloxy, thioaryloxy, thioalkoxy and oxo; each ofA₁, A₂, . . . An−1 and An is independently selected from the groupconsisting of CR″R′″, C═O and C═S, Y is selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, carboxy, saccharide,phosphoric acid, phosphoryl choline, phosphoryl ethanolamine, phosphorylserine, phosphoryl cardiolipin, phosphoryl inositol,ethylphosphocholine, phosphorylmethanol, phosphorylethanol,phosphorylpropanol, phosphorylbutanol, phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine and phsophoglycerol; and each of X₁,X₂, . . . Xn−1 is independently a saturated or unsaturated hydrocarbonhaving the general formula II:

wherein: m is an integer of 1-26; and Z is selected from the groupconsisting of:

whereas W is selected from the group consisting of oxygen, sulfur,nitrogen and phosphor, whereby each of said nitrogen and phosphor issubstituted by at least one substituent selected from the groupconsisting of hydrogen, lone pair electrons, alkyl, halo, cycloalkyl,aryl, hydroxy, thiohydroxy, alkoxy, aryloxy, thioaryloxy, thioalkoxy andoxo; and at least one of X₁, X₂, . . . Xn−1 comprises a Z different thanhydrogen and wherein: each of R₁, R′₁, R₂, . . . Rn−1, Rn, R′n, each ofR″ and R′″ and each of Ra, R′a, Rb, R′b, . . . Rm−1, R′m−1, Rm and R′mis independently selected from the group consisting of hydrogen, a bond,alkyl, alkenyl, alkylnyl, cycloalkyl, aryl, heteroaryl, halo,trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, phosphonate, phosphate, phosphinyl, sulfonyl, sulfinyl,sulfonamide, amide, carbonyl, thiocarbonyl, C-carboxy, O-carboxy,C-carbamate, N-carbamate, C-thiocarboxy, S-thiocarboxy and amino, or,alternatively, at least two of R₁, R′₁, R2, . . . Rn−1, Rn and R′nand/or at least two of Ra, R′a, Rb, R′b, . . . Rm−1, R′m−1, Rm and R′mform at least one four-, five- or six-membered aromatic, heteroaromatic,alicyclic or heteroalicyclic ring; and each of C₁, C₂, . . . , Cn−1, Cn,and each of Ca, Cb, . . . Cm−1 and Cm is a chiral or non-chiral carbonatom, whereby each chiral carbon atom has a S-configuration and/or aR-configuration, a pharmaceutically acceptable salt, a prodrug, ahydrate or a solvate thereof.
 4. The method of claim 3, wherein at leastone of A₁, A₂, . . . and An−1 is CR″R′″.
 5. The method of claim 4,wherein at least one of said at least one of A₁, A₂, . . . and An−1 islinked to a X₁, X₂ . . . or Xn−1 which comprises a Z different thanhydrogen.
 6. The method of claim 3, wherein n equals
 3. 7. The method ofclaim 6, wherein at least one of A₁ and A₂ is CR″R′″.
 8. The method ofclaim 7, wherein A₂ is CR″R′″.
 9. The method of claim 7, wherein each ofA₁ and A₂ is CR″R′″.
 10. The method of claim 6, wherein X₂ comprises a Zdifferent than hydrogen.
 11. The method of claim 10, wherein said Z isselected from the group consisting of


12. The method of claim 11, wherein W is oxygen and each of R″ and R′″is independently selected from the group consisting of hydrogen andalkyl.
 13. The method of claim 12, wherein: each of A₁ and A₂ is CR″R′″;each of B₁, B₂ . . . Bn is oxygen; X₁ is a saturated hydrocarbon havingsaid general formula II, whereas Z is hydrogen; X₂ is a saturatedhydrocarbon having said general Formula II, whereas said Z is

and Y is phosphoryl choline.
 14. The method of claim 13, wherein: X₁ issaid saturated hydrocarbon having said general formula II, whereas Z ishydrogen and m equals 15; and X₂ is a saturated hydrocarbon having saidgeneral Formula II, whereas m equals
 3. 15. The method of claim 13,wherein each of R₁, R′₁, R₂, . . . Rn−1, Rn, R′n, each of R″ and R′″ andeach of Ra, R′a, Rb, R′b, . . . Rm−1, R′m−1, Rm and R′m is hydrogen. 16.The method of claim 15, wherein C₂ is a chiral carbon atom which has aS-configuration or a R-configuration.
 17. The method of claim 15,wherein said oxidized lipid is selected from the group consisting of1-Hexadecyl-2-(4′-Carboxy-butyl)-sn-glycero-3-phosphocholine and3-Hexadecyl-2-(4′-Carboxy-butyl)-sn-glycero-1-phosphocholine.
 18. Themethod of claim 15, wherein said oxidized lipid has the formula:


19. The method of claim 15, wherein said oxidized lipid has the formula:


20. The method of claim 3, wherein n equals
 1. 21. The method of claim20, wherein at least one of R₁ and R′₁ is a phosphate or a phosphonate.22. The method of claim 3, wherein n equals 5 or 6, and at least one ofR₁, R′₁ and at least one of Rn and R′n form at least one heteroalicyclicring.
 23. The method of claim 22, wherein said at least oneheteroalicyclic ring is a monosaccharide ring.
 24. The method of claim3, wherein said oxidized lipid is selected from the group consisting of:1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC),1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC),1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphocholine,1-hexadecyl-2-acetoyl-sn-glycero-3-phosphocholine,1-octadecyl-2-acetoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-butyroyl-sn-glycero-3-phosphocholine,1-octadecyl-2-butyroyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine,1-octadecenyl-2-acetoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-(homogammalinolenoyl)-sn-glycero-3-phosphocholine,1-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-eicosapentaenoyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine,1-octadecyl-2-methyl-sn-glycero-3-phosphocholine,1-hexadecyl-2-butenoyl-sn-glycero-3-phosphocholine, Lyso PAF C16, LysoPAF C18,1-O-1′-(Z)-hexadecenyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine,1-O-1′-(Z)-hexadecenyl-2-oleoyl-sn-glycero-3-phosphocholine,1-O-1′-(Z)-hexadecenyl-2-arachidonoyl-sn-glycero-3-phosphocholine,1-O-1′-(Z)-hexadecenyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine,1-O-1′-(Z)-hexadecenyl-2-oleoyl-sn-glycero-3-phosphoethanolamine,1-O-1′-(Z)-hexadecenyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine,and1-O-1′-(Z)-hexadecenyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine.25. The method of claim 1, wherein said inflammation is associated witha disease or disorder selected from the group consisting of anidiopathic inflammatory disease or disorder, a chronic inflammatorydisease or disorder, an acute inflammatory disease or disorder, anautoimmune disease or disorder, an infectious disease or disorder, aninflammatory malignant disease or disorder, an inflammatorytransplantation-related disease or disorder, an inflammatorydegenerative disease or disorder, a disease or disorder associated witha hypersensitivity, an inflammatory cardiovascular disease or disorder,an inflammatory cerebrovascular disease or disorder, a peripheralvascular disease or disorder, an inflammatory glandular disease ordisorder, an inflammatory gastrointestinal disease or disorder, aninflammatory cutaneous disease or disorder, an inflammatory hepaticdisease or disorder, an inflammatory neurological disease or disorder,an inflammatory musculo-skeletal disease or disorder, an inflammatoryrenal disease or disorder, an inflammatory reproductive disease ordisorder, an inflammatory systemic disease or disorder, an inflammatoryconnective tissue disease or disorder, an inflammatory tumor, necrosis,an inflammatory implant-related disease or disorder, an inflammatoryaging process, an immunodeficiency disease or disorder and aninflammatory pulmonary disease or disorder.
 26. The method of claim 25,wherein said hypersensitivity is selected from the group consisting ofType I hypersensitivity, Type II hypersensitivity, Type IIIhypersensitivity, Type IV hypersensitivity, immediate hypersensitivity,antibody mediated hypersensitivity, immune complex mediatedhypersensitivity, T lymphocyte mediated hypersensitivity, delayed typehypersensitivity, helper T lymphocyte mediated hypersensitivity,cytotoxic T lymphocyte mediated hypersensitivity, TH1 lymphocytemediated hypersensitivity, and TH2 lymphocyte mediated hypersensitivity.27. The method of claim 25, wherein said inflammatory cardiovasculardisease or disorder is selected from the group consisting of anocclusive disease or disorder, atherosclerosis, a cardiac valvulardisease, stenosis, restenosis, in-stent-stenosis, myocardial infarction,coronary arterial disease, acute coronary syndromes, congestive heartfailure, angina pectoris, myocardial ischemia, thrombosis, Wegener'sgranulomatosis, Takayasu's arteritis, Kawasaki syndrome, anti-factorVIII autoimmune disease or disorder, necrotizing small vesselvasculitis, microscopic polyangiitis, Churg and Strauss syndrome,pauci-immune focal necrotizing glomerulonephritis, crescenticglomerulonephritis, antiphospholipid syndrome, antibody induced heartfailure, thrombocytopenic purpura, autoimmune hemolytic anemia, cardiacautoimmunity, Chagas' disease or disorder, and anti-helper T lymphocyteautoimmunity.
 28. The method of claim 25, wherein said cerebrovasculardisease or disorder is selected from the group consisting of stroke,cerebrovascular inflammation, cerebral hemorrhage and vertebral arterialinsufficiency.
 29. The method of claim 25, wherein said peripheralvascular disease or disorder is selected from the group consisting ofgangrene, diabetic vasculopathy, ischemic bowel disease, thrombosis,diabetic retinopathy and diabetic nephropathy.
 30. The method of claim25, wherein said autoimmune disease or disorder is selected from thegroup consisting of chronic rheumatoid arthritis, juvenile rheumatoidarthritis, systemic lupus erythematosus, scleroderma, mixed connectivetissue disease, polyarteritis nodosa, polymyositis/dermatomyositis,Sjogren's syndrome, Bechet's disease, multiple sclerosis, autoimmunediabetes, Hashimoto's disease, psoriasis, primary myxedema, perniciousanemia, myasthenia gravis, chronic active hepatitis, autoimmunehemolytic anemia, idiopathic thrombocytopenic purpura, uveitis,vasculitides and heparin induced thrombocytopenia.
 31. The method ofclaim 25, wherein said inflammatory glandular disease or disorder isselected from the group consisting of pancreatic disease or disorder,Type I diabetes, thyroid disease or disorder, Graves' disease ordisorder, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto'sthyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmuneanti-sperm infertility, autoimmune prostatitis and Type I autoimmunepolyglandular syndrome.
 32. The method of claim 25, wherein saidinflammatory gastrointestinal disease or disorder is selected from thegroup consisting of colitis, ileitis, Crohn's disease, chronicinflammatory intestinal disease, inflammatory bowel syndrome, chronicinflammatory bowel disease, celiac disease, ulcerative colitis, anulcer, a skin ulcer, a bed sore, a gastric ulcer, a peptic ulcer, abuccal ulcer, a nasopharyngeal ulcer, an esophageal ulcer, a duodenalulcer and a gastrointestinal ulcer.
 33. The method of claim 25, whereinsaid inflammatory cutaneous disease or disorder is selected from thegroup consisting of acne, autoimmune bullous skin disease or disorder,pemphigus vulgaris, bullous pemphigoid, pemphigus foliaceus, contactdermatitis and drug eruption.
 34. The method of claim 25, wherein saidinflammatory hepatic disease or disorder is selected from the groupconsisting of autoimmune hepatitis, hepatic cirrhosis, and biliarycirrhosis.
 35. The method of claim 25, wherein said inflammatoryneurological disease or disorder is selected from the group consistingof multiple sclerosis, Alzheimer's disease, Parkinson's disease,myasthenia gravis, motor neuropathy, Guillain-Barre syndrome, autoimmuneneuropathy, Lambert-Eaton myasthenic syndrome, paraneoplasticneurological disease or disorder, paraneoplastic cerebellar atrophy,non-paraneoplastic stiff man syndrome, progressive cerebellar atrophy,Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea,Gilles de la Tourette syndrome, autoimmune polyendocrinopathy, dysimmuneneuropathy, acquired neuromyotonia, arthrogryposis multiplex,Huntington's disease, AIDS associated dementia, amyotrophic lateralsclerosis (AML), multiple sclerosis, stroke, an inflammatory retinaldisease or disorder, an inflammatory ocular disease or disorder, opticneuritis, spongiform encephalopathy, migraine, headache, clusterheadache, and stiff-man syndrome.
 36. The method of claim 25, whereinsaid inflammatory connective tissue disease or disorder is selected fromthe group consisting of autoimmune myositis, primary Sjogren's syndrome,smooth muscle autoimmune disease or disorder, myositis, tendinitis, aligament inflammation, chondritis, a joint inflammation, a synovialinflammation, carpal tunnel syndrome, arthritis, rheumatoid arthritis,osteoarthritis, ankylosing spondylitis, a skeletal inflammation, anautoimmune ear disease or disorder, and an autoimmune disease ordisorder of the inner ear.
 37. The method of claim 25, wherein saidinflammatory renal disease or disorder is autoimmune interstitialnephritis and/or renal cancer.
 38. The method of claim 25, wherein saidinflammatory reproductive disease or disorder is repeated fetal loss,ovarian cyst, or a menstruation associated disease or disorder.
 39. Themethod of claim 25, wherein said inflammatory systemic disease ordisorder is selected from the group consisting of systemic lupuserythematosus, systemic sclerosis, septic shock, toxic shock syndrome,and cachexia.
 40. The method of claim 25, wherein said infectiousdisease or disorder is selected from the group consisting of a chronicinfectious disease or disorder, a subacute infectious disease ordisorder, an acute infectious disease or disorder, a viral disease ordisorder, a bacterial disease or disorder, a protozoan disease ordisorder, a parasitic disease or disorder, a fungal disease or disorder,a mycoplasma disease or disorder, gangrene, sepsis, a prion disease ordisorder, influenza, tuberculosis, malaria, acquired immunodeficiencysyndrome, and severe acute respiratory syndrome.
 41. The method of claim25, wherein said inflammatory transplantation-related disease ordisorder is selected from the group consisting of graft rejection,chronic graft rejection, subacute graft rejection, acute graft rejectionhyperacute graft rejection, and graft versus host disease or disorder.42. The method of claim 25, wherein said inflammatory tumor is selectedfrom the group consisting of a malignant tumor, a benign tumor, a solidtumor, a metastatic tumor and a non-solid tumor.
 43. The method of claim25, wherein said inflammatory pulmonary disease or disorder is selectedfrom the group consisting of asthma, allergic asthma, emphysema, chronicobstructive pulmonary disease or disorder, sarcoidosis and bronchitis.44. The method of claim 1, further comprising administering to saidsubject a therapeutically effective amount of at least one additionalcompound capable of treating or preventing said inflammation.
 45. Themethod of claim 44, wherein said at least one additional compound isselected from the group consisting of a HMGCoA reductase inhibitor (astatin), a mucosal adjuvant, a corticosteroid, a steroidalanti-inflammatory drug, a non-steroidal anti-inflammatory drug, ananalgesic, a growth factor, a toxin, a HSP, a Beta-2-glycoprotein I, acholesteryl ester transfer protein (CETP) inhibitor, a perixosomeproliferative activated receptor (PPAR) agonist, an anti-atherosclerosisdrug, an anti-proliferative agent, ezetimide, nicotinic acid, a squaleninhibitor, an ApoE Milano, and any derivative and analog thereof.
 46. Apharmaceutical composition comprising, as an active ingredient, anoxidized lipid having the general formula I:

wherein: n is an integer of 1-6, whereas if n=1, Cn, Bn, Rn and Y areabsent; each of B₁, B₂, . . . Bn−1 and Bn is independently selected fromthe group consisting of oxygen, sulfur, nitrogen, phosphor and silicon,whereby each of said nitrogen, phosphor and silicon is substituted by atleast one substituent selected from the group consisting of hydrogen,lone pair electrons, alkyl, halo, cycloalkyl, aryl, hydroxy,thiohydroxy, alkoxy, aryloxy, thioaryloxy, thioalkoxy and oxo; each ofA₁, A₂, . . . An−1 and An is independently selected from the groupconsisting of CR″R′″, C═O and C═S, Y is selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, carboxy, saccharide,phosphoric acid, phosphoryl choline, phosphoryl ethanolamine, phosphorylserine, phosphoryl cardiolipin, phosphoryl inositol,ethylphosphocholine, phosphorylmethanol, phosphorylethanol,phosphorylpropanol, phosphorylbutanol, phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine and phsophoglycerol; and each of X₁,X₂, . . . Xn−1 is independently a saturated or unsaturated hydrocarbonhaving the general formula II:

wherein: m is an integer of 1-26; and Z is selected from the groupconsisting of:

whereas W is selected from the group consisting of oxygen, sulfur,nitrogen and phosphor, whereby each of said nitrogen and phosphor issubstituted by at least one substituent selected from the groupconsisting of hydrogen, lone pair electrons, alkyl, halo, cycloalkyl,aryl, hydroxy, thiohydroxy, alkoxy, aryloxy, thioaryloxy, thioalkoxy andoxo; and at least one of X₁, X₂, . . . Xn−1 comprises a Z different thanhydrogen and wherein: each of R1, R′₁, R₂, . . . Rn−1, Rn, R′n, each ofR″ and R′″ and each of Ra, R′a, Rb, R′b, . . . Rm−1, R′m−1, Rm and R′mis independently selected from the group consisting of hydrogen, a bond,alkyl, alkenyl, alkylnyl, cycloalkyl, aryl, heteroaryl, halo,trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, phosphonate, phosphate, phosphinyl, sulfonyl, sulfinyl,sulfonamide, amide, carbonyl, thiocarbonyl, C-carboxy, O-carboxy,C-carbamate, N-carbamate, C-thiocarboxy, S-thiocarboxy and amino, or,alternatively, at least two of R₁, R′₁, R2, . . . Rn−1, Rn and R′nand/or at least two of Ra, R′a, Rb, R′b, . . . Rm−1, R′m−1, Rm and R′mform at least one four-, five- or six-membered aromatic, heteroaromatic,alicyclic or heteroalicyclic ring; and each of C₁, C₂, . . . , Cn−1, Cn,and each of Ca, Cb, . . . Cm−1 and Cm is a chiral or non-chiral carbonatom, whereby each chiral carbon atom has a S-configuration and/or aR-configuration, a pharmaceutically acceptable salt, a prodrug, ahydrate or a solvate thereof, and a pharmaceutically acceptable carrier,the composition being packaged in a packaging material and identified inprint, in or on said packaging material, for use in the treatment orprevention of an inflammation associated with an endogenous oxidizedlipid.
 47. The pharmaceutical composition of claim 46, furthercomprising at least one additional compound capable of treating orpreventing an inflammation associated with an oxidized lipid.
 48. Thepharmaceutical composition of claim 47, wherein said at least oneadditional compound is selected from the group consisting of a HMGCoAreductase inhibitor (a statin), a mucosal adjuvant, a corticosteroid, asteroidal anti-inflammatory drug, a non-steroidal anti-inflammatorydrug, an analgesic, a growth factor, a toxin, a HSP, aBeta-2-glycoprotein I, a cholesteryl ester transfer protein (CETP)inhibitor, a perixosome proliferative activated receptor (PPAR) agonist,an anti-atherosclerosis drug, an anti-proliferative agent, ezetimide,nicotinic acid, a squalen inhibitor, an ApoE Milano, and any derivativeand analog thereof.
 49. The pharmaceutical composition of claim 46,wherein at least one of A₁, A₂, . . . and An−1 is CR″R′″.
 50. Thepharmaceutical composition of claim 49, wherein at least one of said atleast one of A₁, A₂, . . . and An−1 is linked to a X₁, X₂ . . . or Xn−1which comprises a Z different than hydrogen.
 51. The pharmaceuticalcomposition of claim 46, wherein n equals
 3. 52. The pharmaceuticalcomposition of claim 51, wherein at least one of A₁ and A₂ is CR″R′″.53. The pharmaceutical composition of claim 52, wherein A₂ is CR″R′″.54. The pharmaceutical composition of claim 52, wherein each of A₁ andA₂ is CR″R′″.
 55. The pharmaceutical composition of claim 51, wherein X₂comprises a Z different than hydrogen.
 56. The pharmaceuticalcomposition of claim 55, wherein said Z is selected from the groupconsisting of


57. The pharmaceutical composition of claim 56, wherein W is oxygen andeach of R″ and R′″ is independently selected from the group consistingof hydrogen and alkyl.
 58. The pharmaceutical composition of claim 46,wherein n equals
 1. 59. The pharmaceutical composition of claim 58,wherein at least one of R₁ and R′₁ is a phosphate or a phosphonate. 60.The pharmaceutical composition of claim 46, wherein n equals 5 or 6 andat least one of R₁, R′₁ and at least one of Rn and R′n form at least oneheteroalicyclic ring.
 61. The pharmaceutical composition of claim 60,wherein said at least one heteroalicyclic ring is a monosaccharide ring.62. The pharmaceutical composition of claim 57, wherein: each of A₁ andA₂ is CR″R′″; each of B₁, B₂ . . . Bn is oxygen; X₁ is a saturatedhydrocarbon said general formula II, whereas Z is hydrogen; X₂ is asaturated hydrocarbon having said general Formula II, whereas said Z is

and Y is phosphoryl choline.
 63. The pharmaceutical composition of claim62, wherein: X₁ is said saturated hydrocarbon said general formula II,whereas Z is hydrogen and m equals 16; and X₂ is a saturated hydrocarbonhaving said general Formula II, whereas m equals
 3. 64. Thepharmaceutical composition of claim 63, wherein each of R₁, R₁₁, R₂, . .. Rn−1, Rn, R′n, each of R″ and R′″ and each of Ra, R′a, Rb, R′b, . . .Rm−1, R′m−1, Rm and R′m is hydrogen.
 65. The pharmaceutical compositionof claim 64, wherein C₂ is a chiral carbon atom which has aS-configuration or a R-configuration.
 66. The pharmaceutical compositionof claim 64, wherein said compound is selected from the group consistingof 1-Hexadecyl-2-(4′-Carboxy-butyl)-sn-glycero-3-phosphocholine and3-Hexadecyl-2-(4′-Carboxy-butyl)-sn-glycero-1-phosphocholine.
 67. Thepharmaceutical composition of claim 64, wherein said compound has theformula:


68. The pharmaceutical composition of claim 64, wherein said compoundhas the formula: